USE OF THE Mi-1 GENE CARRING TOMATO (Solanum lycopersicum) FOR MANAGEMENT OF ROOT-KNOT DISEASE IN FLORIDA, USA

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1 USE OF THE Mi-1 GENE CARRING TOMATO (Solanum lycopersicum) FOR MANAGEMENT OF ROOT-KNOT DISEASE IN FLORIDA, USA By SILVIA JOÃO SANTOS SÍNEIRO DE OLIVEIRA VAU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017

2 2017 Silvia João Santos Síneiro de Oliveira Vau

3 To my loving parents, João and Silvina Vau whose affection, love, encouragement and prayers provided me with an opportunity to be successful with honor. To the love of my life, best friend, and husband Jonathon Celestine, to my baby Crystal, to my brother and family, Nuno, Teresa, Maria do Mar e João Moisés Vau, to my in-laws Richard and Vicky Celestine, to Robert Celestine and Jackie Bone, and also to my parents from USA and professors Drs. Donald Dickson and Janete Brito, who have been a constant source of knowledge, inspiration and support.

4 ACKNOWLEDGMENTS I would like to express my sincere gratitude to my advisor Professor Donald W. Dickson for the continuous support for my PhD study and related research, for his patience, motivation, and immense knowledge. His guidance helped me during my research and the writing of this thesis. I could not have imagined having a better advisor and mentor for my PhD study. He is indeed the best professor from my entire academic career. I would like to thank other members of my thesis committee: Drs. Jerry Bartz, Janete Brito, and William Crow for their insightful comments and encouragement, but also for the hard questions that provide incentives for me to widen my research from various perspectives. I wish to thank my fellow lab mates for their stimulating discussions, for the sleepless nights we were working together before deadlines and exams, and for all the fun we have had in the last few years. Last but not the least, I would like to thank my family: my parents, my brother, my sister in-law, my nephew, my niece, my in-laws, my brother in-law and husband for supporting me spiritually throughout writing this thesis and my life in general. 4

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS... 4 LIST OF TABLES... 7 LIST OF FIGURES... 9 ABSTRACT CHAPTER 1 INTRODUCTION Root-knot Nematodes (Meloidogyne spp.) Economic Importance Biology and Life History Parasitism by Root-Knot Nematode Root-Knot Nematode Resistant Genes Mi-gene in Tomato Effects of Temperature Virulent Races of Root-Knot Nematodes Tomato Production in Florida Objectives EFFECTIVENESS OF THREE TOMATO CULTIVARS CARRYING THE Mi-1 GENE AND SIX FUMIGANTS FOR MANAGING ROOT-KNOT NEMATODES Introduction Materials and Methods Field Performance of Root-knot Resistant Cultivars Spring Fumigant Effects on Root-knot Nematode Resistance in Tomato Spring and Fall 2013, and Spring Effects of Different Root-knot Nematode Species on Resistant Tomato Spring 2014, 2015, and Fall Results Field Performance of Root-knot Resistant Tomato Cultivars Spring 2012 (Experiment one) Spring 2012 (Experiment two) Fumigant Effects on Root-knot Nematode Resistance in Tomato Spring, Fall 2013, Spring Spring Fall Spring Effects of Different Root-knot Nematode Species on Resistant Tomato Spring

6 Fall Spring Discussion EVALUATION OF THE RESIDUAL EFFECT OF ROOT-KNOT NEMATODE RESISTANT TOMATO CULTIVARS AND FUMIGANTS IN A DOUBLE-CROP SYSTEM Introduction Material and Methods Carrot Trial Cucumber Trial Results Carrot Fall Cucumber spring Discussion CONSTANT AND DIURNAL TEMPERATURE EFFECTS ON THE PENETRATION AND DEVELOPMENT OF ROOT-KNOT NEMATODES (M. arenaria, M. floridensis, and M. javanica) Introduction Material and Methods Nematode Isolates and Inoculum Preparation Plant Material Heat Regime Experiments Data Collection Statistical Analysis Results Discussion LIST OF REFERENCES BIOGRAPHICAL SKETCH

7 LIST OF TABLES Table page 1-1 Host plants reported with resistant genes to root-knot nematodes (adapted from Williamson and Roberts, 2009) Mi gene sources (adapted from: Williamson, 1998; Ammiraju et al., 2003) Effect of soil fumigant treatments and tomato cultivars BHN 602 (susceptible) and Amelia (resistant) on plant vigor, yield and root-knot nematode galling in a field trial on spring 2012 (Experiment 1) Effect of soil fumigant treatment and tomato cultivars BHN 602 (susceptible), Amelia (resistant), and Red Bounty (resistant) on plant vigor, yield, and rootknot nematode galling in a field trial on spring 2012 (Experiment 2) Effect of fumigant treatments and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on spring Effect of fumigant treatment and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on spring Effect of fumigant treatments and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on fall Effect of fumigant treatment and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on fall Effect of fumigant treatments and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on spring Effect of fumigant treatments and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on spring Effect of root-knot nematode species, and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on spring Effect of root-knot nematode species, and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on spring

8 2-11 Effect of root-knot nematode species, and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on fall Effect of root-knot nematode species, and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on fall Effect of root-knot nematode species, and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield s in a field trial on spring Effect of root-knot nematode species, and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on spring Residual effect of fumigant treatments and tomato cultivars in a double crop system with carrot following tomato Residual effect of fumigant treatments and cultivars BHN 602 (susceptible) and Crista (resistant) on second-stage juvenile population densities in soil and the reproductive factor in a double crop system with carrot following Residual effect of fumigant treatments and tomato cultivars in a double crop system where cucumber followed tomato Residual effect of treatment and tomato cultivars BHN 602 (Root-knot nematode susceptible) and Crista (Root-knot nematode resistant) on rootknot nematode population densities in soil and the reproductive factor

9 LIST OF FIGURES Figure page 2-1 Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for spring Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for spring Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for fall Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for fall Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for spring Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for spring Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for fall Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for fall Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for spring Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for spring Photograph illustrating marketable vs. nonmarketable carrot. Nonmarketable carrots were forked, galled, or both symptoms. A) Marketable carrot; B) Nonmarketable carrot. Courtesy of author Mean number of Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days at a constant temperature of 28 C Total number of each Meloidogyne floridensis developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato held 25 days at constant temperature of 28 C

10 4-3 Mean number of Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held 25 days at constant temperature of 28 C Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days at a constant temperature of 28 C Total number of each Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at constant temperature of 28 C Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at constant temperature of 28 C Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days at a constant temperature of 28 C Total number of each Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at constant temperature of 28 C Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at constant temperature of 28 C Mean number of Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 15 and19 days Total number of each Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 15 and 19 days Mean number of Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 19 days at constant temperature of 32 C Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 19 days at a constant temperature of 32 C Total number of each Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held 19 days at constant temperature of 32 C

11 4-15 Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 19 days at constant temperature of 32 C Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 19 and 21 days Total number of each Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivar held for 21 days at constant temperature of 32 C Mean number of Meloidogyne arenaria developmental stages observed in roots of for root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 21 days at constant temperature of 32 C Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 29 days Total number of each Meloidogyne javanica developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 29 days Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 29 days Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 29 days Total number of each Meloidogyne arenaria developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 29 days Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 29 days Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days Total number of each Meloidogyne javanica developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days

12 4-27 Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days at 26 C (12 hours) and 32 C ( Total number of each Meloidogyne arenaria developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days Developmental stages of Meloidogyne floridensis in Agriset 344 roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=late J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4) Developmental stages of Meloidogyne floridensis in Amelia roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=late J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4) Developmental stages of Meloidogyne arenaria in Agriset 344 roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=late J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4) Developmental stages of Meloidogyne arenaria in Amelia roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=J2 egressing from root; Developmental stages of Meloidogyne javanica in Agriset 344 roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4) Developmental stages of Meloidogyne javanica in Amelia roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=late J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4);

13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy USE OF THE Mi-1 GENE CARRING TOMATO (Solanum lycopersicum) FOR MANAGEMENT OF ROOT-KNOT DISEASE IN FLORIDA, USA Chair: Donald W. Dickson Major: Nematology By Silvia João Santos Síneiro de Oliveira Vau August 2017 Tomato (Solanum esculentum L.) is among the most valuable vegetable crops in the United States providing revenues of $2,000,000,000 annually. In warmer climates Meloidogyne spp. can cause heavy suppression of crop yields. The Mi-1 gene in tomato is an effective source of resistance to M. arenaria, M. incognita and M. javanica, however, high soil temperatures affect this resistance. The objectives were to: i) determine the effectiveness of three tomato cultivars carrying the Mi-1 gene and six soil fumigants for managing root-knot nematode species under field conditions, ii) determine the residual effect of root-knot nematode resistant tomato cultivars and fumigants in a double-crop system under field conditions, and iii) compare the effects of constant and diurnal temperatures on the penetration and development of root-knot nematode second-stage juveniles. Based on field experiments there was no indication that daily average temperatures from 20 to 35 C led to the nonfunctioning of tomato cultivars carrying the Mi-1 gene. Nevertheless, in all field trials the yields of root-knot nematode resistant tomato cultivars were consistently less (around 10 to 12%) then from a rootknot nematode susceptible tomato cultivar. However, when the soil was fumigated both cultivars yield similarly. In a double crop system (tomato-carrot, and tomato-cucumber), 13

14 tomato possessing the Mi-1 gene did reduce root-knot nematode population densities in soil; however, there were no significant improvement in yields of carrot or cucumber. When evaluating the development of M. arenaria and M. javanica in environmental controlled chambers set at different temperatures, the resistance in tomato became nonfunctional at 32 C, but not at 28 C. When plants were subjected to a 12 hours. diurnal regime of 26 ºC and 32 ºC the resistance to M. arenaria and M. javanica was maintained. M. Floridensis developed to adult and produced eggs in the resistant tomato cultivar. 14

15 Economic Importance CHAPTER 1 INTRODUCTION Root-knot Nematodes (Meloidogyne spp.) Plant-parasitic nematodes are of great economic importance on vegetable crops. However, because most of them affect plant roots, they represent one of the most difficult disease problems to identify, demonstrate, and manage (Stirling et al., 1998). The detrimental effects of nematodes are commonly underestimated by farmers, agronomists, and disease and pest management consultants. It has been estimated that some 10% of the world s crop production is lost annually as a result of plant nematode damage (Whitehead, 1998). Root-knot nematodes are among the most common and destructive nematode pathogens. They have an extensive host ranges; a high rate of reproduction, and virulence; as well as the ability to interact with other soilborne pathogens in disease complexes (Sasser, 1977). They are able to cause some of the most dramatic above and below ground plant symptoms such as stunted growth, wilting, leaf discoloration and deformation of plant organs. They substantially suppress crop yields by promoting the development of crops with small fruit size, deformation, or physiologic deficiencies e.g., reduction of sugar content induced by Meloidogyne incognita (Kofoid & White, 1919) Chitwood 1949 in sugar beet, and reduction of protein content induced by M. artiellia Franklin, 1961 in legumes (Di Vito and Lamberti, 1977; Sasser, 1980; Di Vito and Greco, 1988). The severity of damage caused by Meloidogyne spp. is often species-specific and varies depending on host, crop rotation, season and soil types (Potter and Olthof, 15

16 1993). Most hosts are considered as susceptible if they support nematode reproduction. Some crop plants may suffer greatly from nematode parasitism and are considered intolerant, whereas others are tolerant because they support nematode reproduction yet produce satisfactory yields. On the other hand, some plants may be infected by the nematode but then activate a defense mechanism that limits nematode development and reproduction, these are referred to as resistant (Perry et al., 2009). Root-knot nematodes are found in all agricultural regions worldwide (Sasser, 1980; Sasser and Carter, 1985). The damage caused by root-knot nematodes tends to be higher in tropical regions than in temperate regions because of greater pathogen diversity, more favorable environmental conditions for pathogen colonization, development, reproduction and dispersal; and the lack of human, technical and financial resources to manage root knot diseases. There are, however, important root-knot nematode species that survive in northern climates, e.g., the northern root-knot nematode (M. hapla Chitwood, 1949). It is the only species that infects carrot as well as other vegetables grown on organic and mineral soils in New York (Mitkowski et al., 2002), northeastern US, and Canada (Belair, 1992). Root-knot nematodes have wide host ranges with over 3,000 reported (Perry and Moens, 2006). Jensen et al. (1977) listed 874 crop species as hosts of seven or eight root-knot nematode species commonly occurring in the Western USA. Although there are more than 100 root-knot nematode species described, the most common and widespread nematode species are: M. arenaria (Neal, 1889) Chitwood, 1949; M. incognita; M. javanica (Treub, 1885) Chitwood, 1949; M. hapla 16

17 Chitwood,1949; M. chitwoodi Golden, O Bannon, Santo & Finley, 1980, and M. fallax Karssen, 1996 (Luc et al., 1990; Evans et al. 1993; Perry et al., 2009). In Florida, there are 14 species reported but the most common on important agricultural crops are M. arenaria, M. incognita, and M. javanica. However, two recently identified species, M. enterolobii Yang & Eisenback, 1983 and M. floridensis Handoo, Nyczepir, Esmenjaud, Van der Beek, Castagnone-Sereno, Carta, Skantar & Higgins, 2004, are being encountered with increased frequency. Each of these species is capable of infecting a large number of important agricultural crops commonly grown in Florida (Sasser, 1977; Moens et al., 2009; Noling, 2015, 2016). Biology and Life History Meloidogyne spp. are obligate sedentary endoparasites. These pathogens establish an intimate relationship with their host plants, inducing the differentiation of parenchyma root cells into multinucleate and hypertrophied feeding cells named giant cells (De Almeida Engler et al., 1999). The successful establishment of feeding cells is essential for nematode development since these cells constitute the exclusive source of nutrients for nematode development (Sasser and Carter, 1985). In addition to the formation of giant cells, the adjoining cortical cells increase in number, referred to as hyperplasia. This increase in cell number results in the formation of galls or knots on infected roots. These root knots are the basis for the nematode s common name. The galls or knots are variable in size and shape depending on the root-knot nematode species and host. For example, M. hapla is particularly known for high incidence of adventitious roots that develop from small root galls (Sasser, 1954), whereas M. trifoliophila Bernard & Eisenback, 1997 in clover produces elongated galls (Mercer et al., 1997). Plants with succulent roots, especially cucurbits and tomato, develop 17

18 relatively large galls, whereas plants with fibrous or woody roots tend to have small or indistinct galls (Perry et al., 2009). The life-cycle of root-knot nematode starts when females lay eggs into gelatinous masses composed of a glycoprotein matrix that is produced by rectal glands in the perineal region of the female (Sharon and Spiegel, 1993). The gelatinous matrix not only gives protection against environmental factors, but also has antimicrobial properties (Orion and Kritzman, 1991). Females may lay 13 to 40 eggs per day, and in the case of favorable hosts and environmental conditions several hundred eggs may be produced by each female (Karssen and Moens, 2006). Within the egg, determinate cleavage and holoblastic embryogenesis proceeds to the development of the first-stage juvenile (J1). The J1 molts within the egg shell to the infective second-stage juvenile (J2) (Perry et al., 2009). The J2 hatches under favorable temperature, moisture, and aeration conditions (Perry, 2002). The J2 using its amphid sensilla, locates a suitable host based on chemical gradients around the roots that are unique in character, concentration, or a combination of both. Although the exact nature of root attractiveness to root-knot nematodes is unclear it is believed that heat, carbon dioxide and plant specific compounds are attractants (Klingler, 1965; Prot, 1980). The only consistent direction of movement seems to be toward carbon dioxide and to the wet end of a soil moisture gradient (Wallace, 1960). There is limited research about repellents or feeding deterrents against nematodes. Some plants do appear to repel or fail to attract nematodes, but there is conflicting evidence about whether it is or is not associated with resistance. Wang et al. (2009) reported that J2 of M. javanica did not have a preference for aggregating or 18

19 penetrating roots of susceptible or resistant cultivars of tomato. However, Griffin (1969) reports that in polyethylene containers the infection of lucerne (Medicago sativa L.) by J2 of M. hapla was significantly higher in susceptible than resistant cultivars. The J2 enters in the zone of root elongation or in areas of lateral root emergence and penetrates by mechanical and chemical action. They move between cortical cells toward the root apex, turn at the meristem, and migrate to the vascular cylinder in the zone of cell differentiation (Starr, 1993; Abad, et al., 2003; Abad and Williamson, 2010). Within 48 to 60 hours after penetration, the J2 stops moving and retardation of normal plant cell differentiation is noticed near their head. The abnormal cells are the beginning of the formation of giant cells, which may reach a final size about 400 times bigger than normal vascular cells. They are not formed by cell wall dissolution, but by inducement of synchronous mitosis without cell division acytokinetic mitosis (Paulson and Webster, 1970; Jones and Payne, 1978; Gheysen and Fenoll, 2002). The nuclei of giant cells are large and have several nucleoli. About 6 days after the J2 infects, the giant cells are filled with a dense cytoplasm (Rohde, 1972; Rohde and McClure, 1975; Wiggers et al., 1990). Giant cell formation requires extensive changes in gene expression. The induction of feeding cells remains poorly understood, but it is reported that effectors secreted by the nematode play a key role in parasitism, with potential direct effects on recipient host cells (Abad et al., 2003; Abad and Williamson, 2010). It is evident that nematode effectors influence auxin transport, plant cell differentiation pathways, inhibition of plant stress and defense responses to establish feeding cells (Urwin et al., 2002; Caillaud et al., 2008; Gheysen and Mitchum, 2011). 19

20 Once a giant cell has been initiated, the J2 becomes sedentary and enlarges assuming a sausage shape. Under favorable conditions, the J2 molts to the thirdstage juvenile (J3), then to the fourth-stage juvenile (J4), and finally the adult stage. The combined time for the J3 and J4 stages transitions is much shorter than that for the J2 or the adult, typically 4 to 6 days. The J3 and J4 lack a functional stylet and do not feed (Perry et al., 2009). Males when present are vermiform and there is no evidence that they feed. Males may be found in parthenogenetic species when conditions are unfavorable for female development, such as overcrowding, limitation of food supply, unfavorable temperatures, presence of resistant hosts, or chemical induction (Castagnone-Sereno, 1993; Castagnone-Sereno, 2006). The majority of root-knot nematodes reproduce by parthenogenesis with only a few exceptions where reproduction is by amphimixis, e.g, M. graminis (Sledge & Golden, 1964) Whitehead, 1968; M. carolinensis Eisenback, 1982; M. microtyla Mulvey, Townshend & Potter, 1975; M. pini Eisenback, Yang & Hartman, 1985; M. megatyla Baldwin & Sasser, 1979; M. spartinae (Rau and Fassuliotis, 1965) Whitehead, 1968, and M. subarctica Bernard, 1981 (Perry et al., 2009). Currently they are considered as minor root-knot nematode species because of their very restricted distribution, host range, and economic impact (Jepson, 1987). Parasitism by Root-Knot Nematode For the completion and success of the root-knot nematode life-cycle a battery of parasitism proteins and effectors are necessary. These includes cell-wall-modifying enzymes of potential prokaryotic origin, multiple regulators of host cell cycle and metabolism, proteins that can localize to the plant cell nucleus, potential suppressors of 20

21 host defense, mimics of plant molecules, and a relatively large cadre of nematode parasitism proteins (Sidhu and Webster, 1981; Davis et al., 2004; 2008). The secreted molecules involved in plant-nematode interactions have their source in several different structures of the nematode. The main secretory organs are amphids, cephalic sensilla, phasmids, esophageal glands, excretory system, and rectum (Eisenback, 1985). Moreover, the cuticle of nematode acts selectively to regulate the flow of fluids though the body wall and, thus, could be a source of secreted compounds recognized as signal molecules by the plant (Abad et al., 2003). An effector secreted by the cuticle of M. hapla is suspected to be responsible for the induction of root hair deformation (Werasinghe et al., 2005). The cuticle also plays an important role as a protective barrier; antioxidant enzymes produced by the hypodermis and secreted though the cuticle may protect the nematode from an oxidative defense response by the host (Robertson et al., 2000). Of the main secretory organs, only the esophageal glands have been extensively studied. Little is known about the molecular structure and role of secretions that may be produced by amphids, cephalic sensilla, phasmids, excretory system, and rectum (Semblat et al., 2001). For instance, amphids and phasmids are chemosensory organs located in the cervical and caudal region, respectively, and are known to be involved with sensing of the environment and releasing signaling molecules that elicit host defenses during the host-parasitic interaction. However, the function of these secretions remain unknown (Caillaud et al., 2008). It is known that esophageal glands produce secretions at different times during parasitism (Neveu et al., 2002). Sub-ventral glands appear to become active and filled 21

22 with vesicles during the migratory phase of parasitism, whereas the dorsal gland only starts an active production of secretions during the sedentary phase of parasitism (Hussey and Mims, 1990). This indicates that sub-ventral and dorsal glands have different roles during parasitism and they need to secrete a complex panel of proteins for the successful establishment of the parasite (Neveu et al., 2002). In research published to date, proteome and transcriptome analysis of esophageal secretions suggest that at least 60 different proteins are potentially produced by esophageal glands during parasitism (Jaubert et al., 2002; Huang et al., 2003). During the migratory phase, sub-ventral glands of J2 secrete several cell wall degrading and modifying enzymes from the stylet. These participate in cell wall softening during penetration and migration in root tissue. The following are compounds believed to be involved in parasitism: beta-1,4-endoglucanases active on cellulose (Rosso et al., 1999; Ledger et al., 2006); pectate lyases and polygalacturonases active on pectin (Jaubert et al., 2002; Huang et al., 2005); xylanases active on hemicellulose (Mitreva-Dautova et al., 2006), and proteins homologous to expansins active on intermolecular bonds between cell wall polysaccharides (Roze et al., 2008). During the sedentary phase, dorsal glands secrete enzymes, unknown molecules, and effectors that help protect root-knot nematode from plant response defenses (Molinari and Miacola, 1997; Gao et al., 2008). The enzyme glutathione S- transferase is assumed to be only involved in the defense of cytoplasmatic reactive oxygen species (ROS) (Dubreuil et al., 2007). The enzyme chorismate mutase is a key enzyme in the shikimic acid pathway directing the synthesis of cellular aromatic amino acids and several secondary metabolites, including phytohormones and plant defense 22

23 compounds. If secreted into the plant cell cytoplasm, the nematode chorismate mutase reduces the synthesis of flavonoids, salicylic acid, phytoalexins, auxin, and phenylpropanoid, which leads to the down regulation of plant defenses (Doyle and Lambert, 2003). The molecule protein plays a crucial role in modulating the signaling events involved in responses to environmental stimuli, progress through the cell cycle and programmed cell death (Takihara et al., 2000). It has been proposed that this molecule interferes with the regulation of cellular stress response and defense, organelle tracking, and the cell cycle in which often acts as an essential interacting protein or chaperone on the plasma membrane of plant cells (Wienkoop and Saalbach, 2003; Jaubert et al., 2004). The molecule calreticulin, a calcium-binding protein, is involved in nuclear export, mrna degradation, cell adhesion, and cell calcium homeostasy during giant cell formation (Kasper et al., 2001). Some nematode effectors that have been identified during giant cell formation include 10A06 (Hewezi et al., 2010), annexins (Patel et al., 2010), SPRY (Rehman et al., 2009; Sacco et al., 2009), and E3-ubiquitin (Bellafiore et al., 2008). However, their role and spatial and temporal delivery into host cells still needs direct experimental evidence (Gheysen and Mitchum, 2011). The complete understanding of the interactions between nematode secretions, and plant signaling and pathways is still a challenge. With the help of genomics, it is expected in the coming years that there will be breakthroughs in understanding the plant nematode interactions from a functional analysis of nematode effector genes as well as plant genes (Gommers, 1981; Davis et al., 2004; 2008). 23

24 Root-Knot Nematode Resistant Genes Meloidogyne spp. are difficult to manage. Frequently large amounts of pesticides must be applied with specialized equipment. With the increase of restrictions on chemical pesticides the role of host resistance for nematode management has grown in importance. Genes conferring resistance against nematodes have been identified in 16 plant species and several of these have been, or are projected to be cloned (Table 1-1) (Williamson and Roberts, 2009). Root-knot nematode resistant traits can vary greatly; resistance can be dominant, recessive, or additive in expression, and can be conferred by a single major gene or by combination of two or more genes, or quantitative trait loci. Plant resistant genes for management of Meloidogyne spp. also differ in their mechanism of action, strength, and durability, and their practical use in plant breeding and nematode management. In general, resistant genes are specific in their elicitation and expression, which make them unique, hard to find, and when found, often times they are not stable because compatible root-knot nematode strains develop quickly (Williamson, 1999). This concept of plant resistance is based on the gene-for-gene interaction where resistant genes (R genes) in the host lead to an incompatible interaction with pathogens possessing the corresponding avirulent genes (Avr gene) (Flor, 1955). The plant response usually involves a rapid disorganization of cell structure (cell membranes and cytoplasm) and function in the infection site. This results in restricting the pathogen s ability to continue their developmental cycle (Trudgill, 1991; Moura et al., 1993). With Meloidogyne spp., resistance in tomato is considered to be the result of a hypersensitive reaction near the head of the J2 that leads to localized cellular necrosis 24

25 (Dropkin, 1969; Paulson and Webster, 1972). The hypersensitive response to an incompatible nematode occurs during the first 12 hours after infection before the J2 establishes a feeding site. It is not related with the physical breakage of cell walls and membranes during the J2 migration, but rather with the secretion of enzymes from the esophageal glands (Paulson and Webster, 1972). The J2 can enter susceptible or resistant tomato plants in about equal numbers (Schneider, 1991; Moura et al., 1993; Windham and Williams, 1994) or in different numbers (Ferris et al., 1982; Lawrence and Clark, 1986; Powers et al., 1992). However, the fate of the juveniles (death in root or emigration) is determined by resistance factors (Williamson, 1999). Several studies showed that infection of roots in resistant tomato cultivars with several species of root-knot nematodes leads to a decrease in the concentration of free phenols (Hung and Rohde, 1973), and an increase in the activity of peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase (Brueske and Dropkins, 1973; Brueske, 1980). The increase in phenylalanine ammonia-lyase activity seems to be directly related with the hypersensitive reaction. Polyphenol oxidases are related with necrotic reactions, and peroxidase activity leads to the abnormal cell wall lignification near the tissue surrounding the necrosis (Goodman et al., 1986). It has been confirmed that ascorbic acid plays an important role in host-parasite interactions by increasing the rate of cyanide-resistant respiration that has an important role in the plant s resistance reaction to root-knot nematodes (Arrigoni et al., 1979). A decrease in ascorbic acid obtained by application of an ascorbic acid synthesis inhibitor led to a reduction of plant resistance to M. incognita, but an artificial increase of ascorbic acid transforms susceptible tomato to resistant ones. 25

26 In general, resistance is supposed to have a significant impact on root-knot nematode reproduction. According with the classical definition of Cook and Evans (1987) resistance is used to describe the ability of the plant to inhibit the reproduction of a nematode species relative to reproduction on a plant lacking such resistance. This implies that a resistant plant should be able to inhibit the reproduction around 90% or more compared with susceptible plants (Robonson, 1969; Taylor and Sasser, 1983). However, resistance to root-knot nematodes does not seem to be consistent. Contradictory research show different results for fecundity of root-knot nematodes between susceptible and resistant plants. Powers et al. (1992) observed more eggs per female of M. incognita and M. arenaria on resistant alyce clover lines (Alysicarpus ovalifolius (Schumach) J. Leonard) when compared to susceptible lines. By contrast, Person Dedryver (1988) showed few eggs per female of M. naasi Franklin, 1965 on resistant ryegrass cultivars (Lolium spp.) when compared with susceptible cultivars; and Anwar and McKenry (2002) observed a complete lack of reproduction of M. arenaria on resistant Vitis spp. The inconsistency of the previous results can be related with the fact that resistance to most root-knot nematodes is effective only against one or a few Meloidogyne spp. and in some cases effective only against certain populations of a species (Williamson and Roberts, 2009). Therefore, continued research and development efforts are needed to exploit transgenic forms of resistance either by using cloned natural resistance genes, such as the use of Mi-gene from tomato, in other Solanaceous spp. crops, or by engineering novel resistance (Goggin et al., 2006). 26

27 Mi-gene in Tomato The first root-knot nematode resistant gene cloned was the Mi-1 from wild tomato, Solanum peruvianum L. (Milligan et al., 1998). This gene was first reported by Smith (1944), and it has been used for more than 63 years (Roberts, 1992). Mi-1 gene confers effective resistance against three root-knot nematode species - M. incognita, M. javanica, and M. arenaria (Williamson, 1998). However, it is not effective against M. hapla, M. enterolobii, or M. floridensis (Brito et al., 2007a, 2007b). The Mi-1 gene was introduced into cultivated tomato, Solanum esculentum Mill, from the wild species by embryo rescue of the interspecific cross (Smith, 1944). However, after Kaloshian et al., 1998 localized the gene on the short arm of chromosome 6 of S. peruvianum, the genetically linked molecular markers: isozyme acid phosphatase and PCR markers were used for introgression instead (Williamson, 1998). The Mi-gene is comprised of several genes: Mi-1 to Mi-9 (Table 1-2). Both Mi-1 and Mi-9 have linked genomic locations (chromosome 6) with numerous homolog clusters, whereas Mi-2 to Mi-8 are independent. For example the gene Mi-3, which imparts resistance to M. incognita and M. javanica populations virulent to Mi-1, is located on tomato chromosome 12, whereas other genes such as Mi-2 are unlinked to both chromosome 6 and 12 (Veremis and Roberts, 1996). From the several Mi-genes available only Mi-1 was incorporated into the domesticated tomato cultivars. Therefore, Mi-1 is currently the best-characterized nematode resistance gene and it serves as a useful basis for comparison with other resistant genes. Several researchers are trying to introduce the Mi-1 gene into other crops such as potato, eggplant and cucumber (Goggin et al., 2004). Historically, a single major 27

28 resistance gene trait has been the focus of most breeding efforts because of their relative ease of manipulation in plant progeny development and selection. Cloning of the Mi-1 gene is important because it would provide a starting point for understanding the basic biology of plant resistance to a parasitic animal and the relationship of Mi-1 gene to other pathogen R-genes (Milligan et al., 1998). DNA sequence analysis of the region of the Mi-1 genome helped to identify three closely related candidate genes: Mi-1.1, Mi-1.2 and Mi-1.3. The functional resistance gene was identified to be Mi-1.2 (Milligan et al., 1998). This gene not only confers resistance to root-knot nematodes, but also to potato aphids (Macrosiphum euphorbiae Thomas, 1878), and whiteflies (Bemisia tabaci Gennadius, 1989) (Rossi et al., 1998; Vos et al., 1995; 1998; Nombela et al., 2003). The Mi-1.2 gene belongs to the group of R genes that contain a leucine-rich repeat region and can be divided into two classes: extracellular with a membrane anchor and cytoplasmatic. The Mi-gene belongs to the group of cytoplasmatic R genes suggesting that the recognition of infective root-knot nematodes by the host is in the cytoplasm (Milligan et al., 1998). The Mi-1.2 gene is constitutive, being expressed in leaves and roots but not in fruit and flowers (Fuller et al., 2008). It is monogenic; nonallelic and the dominant form is homozygous. It is composed of 5,367 bp with two introns and an open reading frame (ORF) of 3,774 bp that encodes a protein of 1,257 amino acids (Chen et al., 2006). It is also composed of a nucleotide binding site (NBS), leucine-rich repeat region (LRR), and lacks a signal sequence. Mi-1 genes have an additional coiled coil (CC) and a leucine zipper (LZ) (Yaghoobi et al., 1995). The leucine-rich repeat region has the major role in determining the specificity of pathogen 28

29 recognition (Ellis et al., 1999); whereas the nucleotide binding site plays an important role on the apoptosis or programmed cell death (Aravind et al., 1999; Hwang et al., 2000); and the leucine zipper and coiled coil allow interaction with other proteins responsible in the recognition of pathogens (Vos et al., 1998). Although the Mi-1 gene is well characterized and its functions defined, little is known about the way the Mi-1 gene is capable of responding to root-knot nematode infection. It may be that Mi-1 is capable of responding to different avirulent products or effectors from root-knot nematodes or a modification of a plant product (Williamson, 1999). Effects of Temperature Temperature can be a major factor that affects the metabolic and developmental rates of nematodes, as well the development of S. esculentum and the functioning of the Mi-1 gene (Abdul-Baki et al., 1996). Certain tomato Mi-genes: Mi-2, Mi-4, Mi-5, Mi-6 and Mi-9 function at temperatures up to 32 C (Table 2); however, Dropkin (1969) reported that resistance conferred by Mi-1 gene is lost at temperatures above 28 C. This is often reported as a limiting factor for implementation of Mi-1 gene in field tomato production. An increase in number of galls has been shown on root-knot resistant tomato cultivars exposed to soil temperatures above 28 C (Holtzman, 1965; Dropkin, 1969; Araujo et al., 1982a; Ammati et al., 1986; Haroon et al., 1993; Wang et al., 2009; Devran et al., 2010). However, there is some discrepancy among these reports. Certain manuscripts have reported the loss of functioning of Mi-1 gene at temperatures of 32 C or below (Dropkin, 1969; Veremis and Roberts, 1996; Williamson, 1998), whereas 29

30 others have shown the Mi-1 gene is still functioning at soil temperatures of 34 C (Ammati et al., 1986; Abdul-Baki et al., 1996; Verdejo-Lucas et al., 2013). This may be related to the number of hours (days) that temperatures are above 28 C and (or) the sequence of heat and timing of inoculation. Dropkin (1969) reported that at a constant temperature of 28 C only 2% of M. incognita J2 developed within the roots of resistant tomato in contrast with 87% at 33 C. Similar results were obtained for M. arenaria and M. javanica at 30 and 32 C. He also found that elevated temperatures early in the infection process determined the course of M. incognita development in a tomato cultivar containing the Mi-1 gene. Plants that were inoculated and maintained at 32 C for 1 to 3 days, and then subsequently held at 27 C for 1 month, contained abundant galls and eggs when compared with plants inoculated and maintained for 2 days at 28 C and then subsequently held at 32 C for 1 month. Recently it was reported that a heat treatment of 35 C for 3 hours before inoculation followed by a constant temperature of 25 C was enough to increase the number of galls of M. incognita on a resistant tomato cultivar containing the Mi-1 gene (Carvalho et al., 2015). Oddly, the number of galls per plant with a treatment of 6 days at 35 C for 3 hours before inoculation followed by a constant temperature of 27 C did not differ from that obtained when plants were held at a constant temperature of 25 C. It was suggested that Mi-1 gene resistance either acclimates to or recovers from exposure to high temperatures. This hypothesis is supported by Zachero et al. (1995) who reported the recovery of resistance at 27 C after 6 days of exposure to a constant temperature of 34 C. They also found that 2 days at 34 C were required for increase susceptibility of cv. VFN8 to M. incognita; and the loss of susceptibility was 30

31 accompanied by an increase of peroxidase activity. However, Araujo et al. (1982b) showed that the longer the initial incubation period was maintained at high temperatures (32.5 C) followed by a subsequent period of low temperature (25 C), resulted in an increase in the number of egg masses per plant root system. Similar results were reported when the inoculation of tomato plants with J2 was done before the heat treatment instead of after (Carvalho et al., 2015). Studies indicate that resistance to M. incognita breaks down at temperatures equal to or higher than 32 C or 34 C; however, these studies employed heat treatments of no more than 24 hours (Abawi and Barker, 1984; Devran et al., 2010; Veremis and Roberts, 1996; Zacheo et al., 1995). Verdejo-Lucas et al. (2013) found that Mi-1 gene resistance to M. arenaria and M. javanica remained intact in three of five resistant tomato cultivars exposed for 65 days to ambient soil temperatures as high as 34.1 C for more than 7.5 hours per day. These studies suggest that tomato maintains its resistance when exposed to daily temperature fluctuations. Also, Araujo et al. (1982a) showed that plants exposed to differential low nocturnal temperatures (19 to 25 C) and high diurnal temperatures (31 C) were better able to maintain resistance than plants exposed to a constant high temperature. It is important to understand the influence of thermoperiodism on the functioning of the Mi-1 gene, particularly studies that are able to simulate the temperature effects over an entire tomato growing season. In addition to treatment timing, duration, and nematode exposure to heat and nematode species; differences in the tomato genotype are also influenced by temperature. Variation in Meloidogyne spp. reproduction on resistant tomato genotypes has been attributed to the plant s genetic background, level 31

32 of zygosity, and possibly an additional locus or other factor in the resistant genotype affecting resistance expression (Abdul-Baki et al., 1996; Jacquet et al.; 2005). Homozygous dominant plants (Mi:Mi) are less susceptible to galling (Barham and Winstead, 1957; Laterrot, 1973) and nematode reproduction than heterozygous plants (Jacquet et al., 2005), which suggest an additive gene action at the Mi locus (Abdul- Baki and Haroon; 1996). However, Abdul-Baki et al. (1996) showed that homozygous or heterozygous genotypes of tomato were equally resistant to M. incognita and M. arenaria. Virulent Races of Root-Knot Nematodes In addition to heat instability of the Mi-1 gene, virulent populations of Meloidogyne spp. exist that are capable of overcoming this resistance. They can occur naturally without previous exposure to or selection by tomato with the Mi-1 gene (Netscher, 1976; Prot, 1984), or by the continuous or repeated planting of resistant tomato cultivars (Roberts, 1992). Certain populations of M. incognita and M. javanica have been reported to infect and to reproduce on Mi-1 tomato cultivars all over the world including: California (USA) (Kaloshian et al., 1996); Cyprus (Philis and Vakis, 1977); France (Castagnone-Sereno et al., 1994; Jarquin-Barberena et al., 1991); Greece (Tzortzakakis et al., 2005; Tzortzakakis and Gowen, 1996); Israel (Iberkleid et al., 2014); Italy (Molinari and Miacola, 1997); Morocco (Eddaoudi et al., 1997); and Spain (Ornat et al., 2001). Previous field trials have demonstrated the capacity of certain species or races of root-knot nematodes to reproduce and inflict damage upon resistant tomato cultivars (Cortada et al., 2008; Verdejo-Lucas and Sorribas, 2009; Verdejo-Lucas et al., 2009). Given that significant yield suppression can still occur, combined efforts to manage soil 32

33 population densities to low levels before planting should be considered. If this situation develops, the use of a nematicide and resistant cultivar may be needed to provide adequate results (Roberts et al., 1986). Because Florida tomato growers relied on methyl bromide for more than 45 years, there was little or no interest in growing root-knot nematode resistant tomatoes. This is in contrast to California were a majority of field-grown processing tomatoes have the Mi-1 gene (Cook, 2004; Williamson and Roberts, 2009). Nevertheless, with the phase out of methyl bromide, and the lack of effective and environmental friendly chemical alternatives to manage root-knot nematodes, Florida growers may be more apt to use root-knot nematode resistant tomato cultivars in the future (Rich and Olson, 1999; 2003; Rich et al., 2003). Tomato Production in Florida The United States is one of the world's leading producers of tomato behind China and India. Fresh and processed tomato account for more than $2 billion in annual farm cash receipts (Zhu et al., 2013). Fresh-market tomato is produced in every state in the nation, with commercial-scale production in about 20 States. National fresh-market tomato acreage has been trending lower over the past several decades, mainly due to the imports of tomato from Mexico (Zhu et al., 2013). Mexico s competitive advantages in production costs and favorable government policies resulted in Mexico becoming the leading country for tomato exports. Since implementation of the North American Free Trade Agreement (NAFTA) in 1994, tomato exports from Mexico to the USA have increased from 2.4 billion pounds in 2009 to 3 billion in Meanwhile US tomato production fell from 1.55 billion pounds in 2005 to 0.96 billion pounds in 2012, a drop of 40% (Zhu et al., 2013). 33

34 The top two fresh-market tomato production states are Florida and California. Together these states produce fresh-market tomato on 12,141 to 16,187 hectares, which represents almost two-thirds of total US fresh-tomato hectares (a share that has not changed much since the 1960s). Ohio, Virginia, Georgia, and Tennessee round out the top six states in terms of area planted (Anonymous, 2014). Tomato production in Florida began in Alachua County in 1872 with a large scale planting located at Palmer, FL (Weber, 1939; Crill et al., 1977). Since then, Florida had continued to provide fresh market tomatoes and ranks first in the nation in value of the crop. California is the leading state for producing processed tomato. Tomato is the most popular vegetable, or fruit, in the United States. The Florida vegetable industry grew because of the demand for fresh produce in the US, especially during the winter months (Rose, 1973). Advantages to tomato production in south Florida include its subtropical climate, with mild temperatures throughout the winter and mid-season periods of rainfall (Pohronezny et al., 1986). Based on data from 2001, the principal tomato production regions in Florida are partitioned into five districts: Dade County, east coast (Broward, Palm Beach, Martin and St Lucie Counties), southwest (Collier, Lee and Hendry Counties), Tampa Bay area (Sarasota, Manatee, Hardee and Hillsborough Counties), and northwest (Gadsden County) (Mossler et al., 2015). In Florida, tomato is commercially grown on soils that are considered infertile. In addition, these soils are often infested with soilborne pathogens including nematodes. The primary nematode parasites of tomato includes root-knot nematodes and sting nematodes (Belonolaimus longicaudatus Rau, 1958); either of which may cause extensive root damage and yield suppression (Overman et al., 1965). Tomato 34

35 production problems on the sandy soils of Florida often involve complexes of nematodes, soil insects, and plant disease, such as fungal wilts, fungal blights and damping-off, and bacterial wilt (Powell, 1971). Many factors influence nematode damage and their interaction with other soilborne pests. Broad-spectrum soil fumigants have been used to alleviate the severity and recurring nature of pathogens and pests (Jones and Overman, 1976). For around 45 years methyl bromide (Mbr) formulated with varying amounts of chloropicrin was applied in 93% of Florida tomato acreage. Between 2.4 to 3.7 million kilograms of active ingredient were applied each year (Bloem and Mizell, 2004). Beginning in 1993, as a result of signatory of the Montreal Protocol, the Environmental Protection Agency (EPA) mandated the phase out of Mbr for soil fumigation in the U.S. by the year The use of Mbr continued until 2014 on a more limited basis based on critical use exemption request (Anonymous, 2017). Growers are corrently using different chemical and non-chemical approaches as alternatives to Mbr. In a survey conducted by the USDA, 70% of growers were using 1,3-dichloropropene (1,3-D) in combination with chloropicrin (Pic) as an alternative to Mbr. From that percentage of growers, 60% indicated they would try metam sodium (metam Na) or potassium (metam K) as an alternative. In general, 55% of the growers would try to rotate crops, 30% would apply only Pic, and 15% would try soil solarization as a means to manage pathogens and pests formerly managed with Mbr. Where double cropping is practiced, data showed that pathogens or pest densities were higher following alternatives other than following Mbr. This suggests that it will be more difficult to economically produce a second crop without Mbr (Anonymous, 2014). 35

36 The use of Mi-1 gene tomato as a rotation crop, in combination with existing and new nematicide chemistry might be a good alternative for tomato production in Florida (Rich and Olson, 2003). Little or no research has been conducted in Florida to evaluate field grown tomato cultivars containing the Mi-1 gene with the exception of Gadsden County in the North Florida production area (Rich and Olson 2004). It is important to evaluate the effectiveness of tomato cultivars containing the Mi-1 gene in the other tomato growing regions mentioned above. Objectives The objectives of this project were to: (1) determine the effectiveness of three tomato cultivars carrying the Mi-1 gene and six fumigants for managing root-knot nematodes species under field conditions; (2) determine the residual effect of root-knot nematode resistant tomato cultivars and fumigants in a double-crop system under field conditions; and (3) compare the effect of constant and diurnal temperatures on the penetration and development of different root-knot nematode species (M. arenaria, M. enterolobii, M. floridensis, M. incognita, and M. javanica). 36

37 Table 1-1. Host plants reported with resistant genes to root-knot nematodes (adapted from Williamson and Roberts, 2009). Host plant Resistance gene or source Meloidogyne species References Alfalfa Mj - 1 M. hapla (Mh), M. incognita (Mi) Potenza et al., 1996; 2001 Carrot Mj - 1 Mi, M. javanica (Mj) Boiteux et al., 2001 Clover TRKR M. trifoliophila Mercer et al., 2005; Mercer, 2005 Coffee Mex - 1 M. exigua Anthony et al., 2005 Common me 1, me 2, me 3 Mh, Mi, Omwega and Roberts, bean Mj 1992 Chen and Roberts, 2003 Cotton Rkn 1, RKN 2 Mi Wang et al., 2006; Wang et al., 2008 Cowpea RK, Rk2, rk3 M. arenaria (Ma), Mh, Mi, Mj Roberts et al., 1996; Ehlers et al., 2000 Grape N, Mur1 Ma, Mi Cousins et al., 2003 Lima bean Mir-1, Mig-1, Mjg-1 Mi, Mj Roberts et al., 2008 Groundnut Arachis spp. hybrids Ma, Mj Choi et al., 1999; Church et al., 2005 Pepper Me1, 3,4,7; Mech1,2 Ma, Mi, Mj, M. chitwoodi (Mc) Djian-Caporalino et al.,

38 Table 1-1. Continued. Host plant Resistance gene or source Meloidogyne species References Potato Rmc1, MfaXIIspI Mc, Mh, M. fallax (Mf), Mi Brown et al., 1996; Janssen et al., 1997; Kouassi et al., 2006 Prunus Ma Mj, Mi, Ma Dirlewanger et al., 2004 Soybean 2 QTLs Mj Tamulonis et al., 1997 Sugarbeet Beta vulgaris spp Ma, Mc, Mh, Mf Yu et al., 2001 Sweet potato Poorly understood* Mi, Mj, Ma Jones and Dukes, 1980 Tobacco Rk Mi Yi et al., 1998 Tomato Mi-1 Mi-9 Mi, Mj, Ma Yaghoobi et al., 1995 Veremis and Roberts, 1996, Ammiraju et al., 2003 Wheat Triticum tauschii Mi, Mj, M. chitwoodi Kaloshian et al., 1991 *The genetics of resistance to root-knot nematodes in sweet potato is poorly understood and the research is contradictory (Cordner et al., 1954; Giamalva et al., 1961; Jones and Dukes, 1980; Struble et al., 1966; Ukoskit et al., 1997). 38

39 Table 1-2. Mi gene sources (adapted from: Williamson, 1998; Ammiraju et al., 2003). Gene Source Properties Genetics Mi (Mi-1) Solanum peruvianum Resistant to: M. arenaria; M. Mapped to short arm of PI incognita, M. javanica; resistance lost chromosome 6; cloned at >28 C Mi 2 PI R2 Resistant to M. incognita up to 32 C Resistant to: M. arenaria; M. Not linked to Mi or Mi-3, linked to Mi-8 incognita, M. javanica, M. hapla Mi 3 PI MH Resistant to Mi-virulent M. incognita 557R Mapped to short arm of chromosome 12; linked to Mi-5 Mi 4 LA Resistant to M. incognita and M. javanica up to 32 C Specific clone from an accession line Mi 5 PI MH Resistant to M. incognita and M. javanica up to 32 C Linked to Mi-3, linked on chromosome 12 Mi 6 P MH Resistant to M. incognita up to 32 C Linked to Mi-7 Mi 7 PI MH Resistant to Mi-virulent M. incognita Linked to Mi-6 557R up to 25 C 39

40 Table 1-2. Continued. Gene Source Properties Genetics Mi 8 PI R2 Resistant to Mi-virulent M. incognita Linked to Mi-2 557R up to 25 C Mi 9 LA2157 Resistant to M. arenaria; M. incognita, M. javanica up to 32 C Mapped to short arm of chromosome 6 40

41 CHAPTER 2 EFFECTIVENESS OF THREE TOMATO CULTIVARS CARRYING THE Mi-1 GENE AND SIX FUMIGANTS FOR MANAGING ROOT-KNOT NEMATODES Introduction Florida is the leading fresh-market tomato producer in the USA. One of the major reasons is related with the fact that per year, tomato is grown consecutively during the winter months (Rose 1973). Southern Florida s climate is subtropical, with mild temperatures through the winter and with seasonal periods of high rainfall that allows for vegetable production. Cropping patterns are reversed from those in temperate regions. In Florida there are generally two crops, although in the southern most regions plantings can be continuous from September to January with no discernible fall-spring dichotomy. In general, planting dates in south Florida are August to January, in central Florida August to September and January, and in north Florida July to August for fall and February to March for spring plantings (Schuster et al., 1996). The production system used by Florida tomato growers generally includes soil fumigation, polyethylene film mulched beds, and drip or seep irrigation. Almost 100% of the crop is grown on raised beds covered with film (Schuster et al., 1996). Raised beds permit the formation of a more defined rooting zone and improves drainage during high rainfall periods (Jones et al., 1991). The height and width of beds vary, as does the type of film and irrigation regimes (drip or seep irrigation). There are several types of films including black, white, white-on-black, gray, reflective, or films that can be total impermeable (TIF) or virtually impermeable (VIF). In a 2012 survey 51% of tomato growers were using VIF because of its effectiveness in weed management, particularly annual weeds, retention of fumigants, and because it meets the requirements of the new rules and regulation on fumigant applications (Zhu et 41

42 al., 2013). In general, the use of film improves crop response because it modifies soil temperature, preserves soil moisture, minimizes nutrient leaching during heavy rainfalls, maintains bed integrity, reduces incidence of fruit rot, and decreases fumigants volatilization (Jones et al., 1991). The selection of film type is related with the growing season, type of fumigants applied, or with the management of certain pests. Black films are used during spring seasons to increase soil temperature, whereas white or whiteon-black films are used in fall seasons to keep soil temperature low. Reflective metallic aluminum coated films are used to reduce the incidence of thrips, whiteflies and aphids (Antignus et al., 1998). Bedding is accomplished using a bed press, bedding disks or double-disk hillers, followed by a board to level the bed tops. Fertilizer is applied in bands, injected within the irrigation system, or broadcast before planting. Soil fumigants are applied before the film is applied, or simultaneously via a bedder applicator. Bed widths vary but the most common is 81 centimeters and plant spacing is generally 45 centimeters. Beds are centered at 1.8 meters (Hochmuth et al., 1998). Tomato seedlings are generally transplanted 3 to 4 weeks after fumigation. The seedlings are staked and tied with tomato string two to three times during the growing season. Generally around 12,000 plants per hectare are transplanted (Hochmuth and Vavrina, 1997). Most tomato cultivars require 80 to 90 days from transplanting to the final harvest. Depending on the market, tomato fruit may be picked two to three times during the season. After final harvest, plants may be killed with a burn down herbicide. Historically, tomato growers have relied on methyl bromide (Mbr) as a broadspectrum fumigant to reduce the incidence of soilborne pests, pathogens and weeds 42

43 (Gilreath et al., 1994). With the Montreal Protocol and Clean Air Act, the Environmental Protection Agency (EPA) mandated a phase out of Mbr for However, the tomato industry was granted critical use exemptions for limited use of Mbr through the end of Several alternative fumigants have been developed and trialed with some success. However there is no single alternative that provides the same degree of effectiveness previously provided by Mbr. The most notable limitation of alternative chemistries is the longer re-entry and plant back intervals, and the lack of weed management. For example, data from grower s surveys indicate that to achieve satisfactory tomato yields the application of different fumigants (i.e., 1,3-dichloropropene (1,3-D) and chloropicrin (Pic), plus a separate complementary herbicide treatment are required (Aerts, 1999). Currently the fumigants that are most commonly suggested as alternatives to Mbr are: 1,3-D, methyl isothiocyanate generators, Pic, and dimethyl disulfide (DMDS). Several growers are currently applying a combination of 1,3-D, metam sodium or potassium and Pic. The fumigant nematicide 1,3-D was introduced by Dow AgroSciences, LLC in 1956 and currently sold under the trade name Telone II (96% 1,3-D). The 1,3-D is also formulated with Pic, which serves as a multi-purpose material for nematode and fungal pathogen management. These combined formulations include: Telone C-17 (81% 1, 3- D and 17% Pic) and Telone C-35 (63% 1,3-D and 35% Pic). The fumigant 1,3-D provides a relatively inexpensive means for nematode management. In fresh-market tomato, previous studies have shown that numbers of stunt (Tylenchorhynchus spp.), 43

44 sting (Belonolaimus spp.), stubby-root (Trichodorus spp.), and root-knot (Meloidogyne spp.) nematodes and the damage they cause are reduced with in-bed soil injections of 1,3-D + Pic (Gilreath et al., 2004a, 2004b, 2005a, 2005b, 2006a). The studies of Dickson et al. (1998) and Mirusso et al. (2002) determined that there were no differences in root galling caused by root-knot nematodes following treatments with 1,3- D + Pic and treatments with MBr + Pic. Similar results were reported in tomato by Eger (2000), Locascio and Dickson (2000), and Locascio et al. (2001). Weed management, however, remains a challenge in Florida tomato production. Weed species regularly occurring in Florida tomato production fields includes crabgrass (Digitaria spp.), bermudagrass (Cynodon spp.), barnyardgrass (Echinochloa spp.), goosegrass (Eleusine spp.), fall panicum (Panicum spp.), pigweed (Amaranthus spp.), common purslane (Portulaca spp.), common lambsquarters (Chenopodium spp.), nightshade (Solanum spp.), pusley (Richardia spp.), eclipta (Eclipta spp.), sida (Sida spp.), evening primrose (Oenothera spp.), and beggarweed (Desmodium spp.). These weeds usually grow in row-middles and through planting holes within the polyethylene film. In contrast, the sedges - purple (Cyperus rotundus L.) and yellow (C. esculentus L.) penetrate the polyethylene film, making these species especially difficult to manage. The methyl isothiocyanate-generating materials, namely metam sodium and metam potassium provides some management of weeds and fungal pathogens, however, these materials are limited in their applicability as stand-alone fumigants as they are highly variable in their effectiveness on root-knot nematodes. Chloropicrin is considered as an excellent soil fungicide. It has been shown to have some nematicidal activity, although it is not as effective as other existing soil 44

45 fumigants, such as 1,3-D. However, its contribution for pathogen control is significant, and the majority of studies include Pic as a component in a multi-tactic approach. Dimethyl disulfide is a recently labeled broad-spectrum soil fumigant from Arkema, Inc. It was registered in Florida in 2011 for pre-plant application to manage soilborne pathogens and pre-emergent weeds. Field treatment results are promising, however, two negative aspects need to be taken into consideration: the control of some weeds, particularly grasses, seems to be poor and in some situations DMDS promotes their growth; and the large buffer zones required because of its pungent sulphur odor (unpubl. data). Since the number of efficient chemical alternatives to Mbr is few, it is necessary to look at different approaches for the suppression of nematodes in tomato fields. One of the most promising management tactics is crop host resistance. Numerous tomato cultivars are available that contain the Mi-1.2 gene that suppresses the reproduction of root-knot nematodes, namely Amelia cv., Brixmore cv., Crista cv., Fletcher cv., Marianna cv., Monticello cv., Red Bounty cv., Sanibel cv., Santa cv., Shiren cv, Sunoma cv., and Tachi cv. None of these have been extensively tested under field conditions in Florida, especially under high root-knot nematode pressure. In Florida the first research comparing yields and the degree of root-knot nematode galling on resistant versus susceptible tomato cultivars was done in 1999 (Rich and Olson, 1999). The presence of two root-knot nematode species with the potential to break resistant genes, particularly Mi-1 gene: M. enterolobii, and M. floridensis have been reported in Florida. No studies have been done to evaluate the performance of resistant cultivars under field conditions (Brito 2007a; 2007b). 45

46 Therefore, more research is necessary to evaluate the performance of root-knot nematode resistant tomato cultivars under field conditions. The objectives were (i) to evaluate the performance of tomato cultivars containing Mi-1.2 gene vs. susceptible cultivars, (ii) to determine the response of resistant and susceptible cultivars used in combination with fumigants, and (iii) to determine effectiveness of resistant tomato where populations of M. enterolobii and M. floridensis exist. Materials and Methods Field Performance of Root-knot Resistant Cultivars Spring 2012 Two field experiments were conducted at the University of Florida Plant Science Research and Education Unit, Citra, FL (PSU). The soil was Arredondo fine sand with 95% sand, 3% silt, and 2% clay; organic matter 1.5%, 1 to 2 meters in thickness, with underlying loamy material; and ph 6.5. The field site was infested with a mixture of Meloidogyne incognita, M. javanica, and M. arenaria. The population densities were increased by planting okra (Hibiscus esculentum L. cv. Clemson Spineless) across the experimental field site during the previous season. There were moderate to heavy infestations of both purple and yellow nutsedges, and Florida and Brazilian pusley. In preparation for tomato transplant, the field site was disked, plowed and cultivated in mid-february. Before bedding a starter fertilizer (N-P 2 O 5 -K 2 O) plus minors was applied at a rate of 1,613 kg/ha and incorporated immediately before bed formation and fumigant application. Raised beds were formed with a Kennco powerbedder (Kennco Mfg., Ruskin, FL). Immediately after bed formation fumigant treatments that included 1,3-D, or 65% 1,3-D + 35% Pic at the rates of 140 l/ha and 325 l/ha, respectively, were injected at 25 cm deep, with three swept-back chisels equally spaced over the bedtop with a film-laying fumigant applicator (Kennco mini-combo, 46

47 Ruskin, FL). Nitrogen gas was used to pressurize the fumigant cylinder. The polyethylene film and drip tube was laid as the fumigant was applied. The beds were 23-cm tall, 91-cm wide, 12-m long, and spaced on 1.8 m centers. The film used was black, low density polyethylene (1.25 mil thickness). A single drip tube (8 mil, 30-cmemitter spacing) with a flow rate of 1.9 l/min/30.5 m of row (Chapin Irrigation Products, San Marcos, CA) was placed ca. 5-cm deep along the length of the row and ca. 12 cm off center under the film as it was applied. After a waiting period of 22 days (mid-march 2012) tomato cvs. Amelia and Red Bounty (resistant) and BHN 602 (susceptible) were transplanted in the bed with a plant spacing of 45 cm. For both experiments, treatments were arranged in a randomized complete block design with six treatments and six replications. Experiment one consisted of two cultivars Amelia (resistant) and BHN 602 (susceptible) and two fumigants 1,3-D and 65% 1,3-D + 35% Pic, and a nontreated control. Experiment two consisted of three cultivars Amelia, Red Bounty (resistant), and BHN 602 (susceptible), and one fumigant treatments 65% 1,3-D + 35% Pic, and a nontreated control. Nitrogen and K 2 O were applied weekly via drip irrigation for a total of 8 to 10 weeks. The fertilizer was formulated from 42% urea and 62% potassium chloride. A total of 225 kg/ha for N and 202 kg/ha for K 2 O were applied over the entire crop season. After beds were covered with the polyethylene film the row middles were treated with a mixture of metribuzin, napropamide, halosulfuron-methyl, metalochlor, and trifluralin for weed control. Immediately following transplanting, dinotefuran was applied through the drip tube to suppress white flies, and granular chloropyrifos insecticides was broadcast 47

48 over the beds to suppress cutworms and mole crickets. Foliar fungal pathogens were managed by twice weekly alternating spray applications of chlorothalonil, mancozeb and azoxystrobin. All tomato plants were staked and tied with tomato string during the growing season. Plant vigor was evaluated 2 weeks before harvest. The rating was based on a subjective scale of one to 10; with one = small, chlorotic and non-vigorous plants and 10 = large, dark green and vigorous plants. Fruit were handpicked at the breaker stage and again 12 days later. The fruit was graded to size (Kerian Sizer, Kerian Machines, Grafton, ND) of extra-large, large, medium, and culls. The first three categories were weighed and marketable yield was recorded. After the final harvest, 10 root systems (arbitrarily chosen) per plot were subjected to a root-gall rating based on a zero to 100% scale where zero = no visible galls, 10 = 10% of root system galled, and 100 = 100% of the root system galled (Barker et al., 1986). Post-plant nematode population densities in soil were assessed by taking six soil cores per plot to a depth of ca. 20 cm using a 2.5-cm-diam cone-shaped soil probe. Nematodes were extracted from a 100-cm 3 subsample using the centrifugal-flotation technique (Jenkins, 1964). All plant-parasitic nematodes were counted by observation with an inverted microscope at x 40. All data were subjected to analysis of variance (ANOVA) for a complete block design and means were compared by Duncan s new multiple-range test (SAS Institute, Cary, NC). Unless otherwise stated all differences listed in the results are at the P 0.05 level. 48

49 Fumigant Effects on Root-knot Nematode Resistance in Tomato Spring and Fall 2013, and Spring 2014 Two experiments in 2013 (spring and fall), and one experiment in spring 2014, were conducted comparing the root-knot nematode resistance in Crista vs. the susceptible BHN 602 when soil was fumigated with different fumigants. The methods used in these three experiments were the same as that mentioned for experiment 1, except that a white on black low density polyethylene film was used for the fall crop. The fumigants evaluated in spring 2013 included: DMDS (374 l/ha); metam K (561 l/ha); Mbr 50% and Pic 50% (393 kg/ha); Pic (263 kg/ha); 1,3-D (140 l/ha); and 65% 1,3-D + 35% Pic (325 l/ha). Treatments were arranged in a randomized split-plot design with six treatments and five replications. Fumigants and a nontreated control comprised the main-plots, and cultivar types comprised the sub-plots. The harvest of tomato fruit followed the same methods mentioned for experiment 1. Root galling and final nematode population densities were assessed in late June for the spring crop and late November for the fall crop. For all trials soil temperature under the polyethylene film was measured at three depths: surface, 8, 15 and 23 cm deep in two beds, chosen arbitrarily, using a data logger (StowAway TidBit, Onset Computer Corp., Bourne, MA). The temperature was recorded every 15 minutes from the date of transplanting to final harvest. Daily average temperatures were calculated based on the equation: (maximum temperature + minimum temperature 2); and the daily heat units were calculated based on the sum of minutes with temperatures above 28 C. 49

50 All data were subjected to analysis of variance (ANOVA) for split-plot design and means were separated using Tukey s honest significant difference (HSD) (SAS Institute, Cary, NC). Unless otherwise stated all differences listed in the results are at the P 0.05 level. Effects of Different Root-knot Nematode Species on Resistant Tomato Spring 2014, 2015, and Fall 2014 Two experiments in 2014 (spring and fall) and one experiment in spring 2015, were conducted. The procedures mentioned above for field preparation and transplanting of tomato cultivars were similar as that for experiment 1. Five treatments were evaluated, and the cultivars used were Crista and BHN 602. The treatments included root-knot nematode species each inoculated in individual plots in the spring and fall of The species included M. arenaria race 1, M. enterolobii, M. floridensis, M. javanica, and M. incognita race 2. Before the different species were added to soil an application of 1,3-D at a rate of 140 l/ha was applied broadcast over the area 1 month before inoculation. All species were derived from a single egg mass isolate. They were increased on tomato cv Agriset 334 in a greenhouse at the Entomology and Nematology Department, University of Florida. Species and race identification were confirmed using perineal patterns, isozyme phenotypes, and differential host tests (Esbenshade and Triantaphyllou, 1990; Harris and Hopkinson, 1976; Hartman and Sasser, 1985).The inoculum for field application was prepared by extracting eggs from galled tomato roots that were immersed in 0.5% NaOCl solution (Bonetti and Ferraz, 1981). The eggs were caught on a sieve with 25-µm-pore openings, and the concentration of eggs and juveniles was estimated using a Peters counting slide (Peters, 1952). The volume of 50

51 suspension was adjusted with tap water to obtain a concentration of 1,000 eggs and juveniles/ml. Approximately 5,000 eggs and (or) juveniles were added by pipetting separately into 12 plant-holes that were pre-punched 46 cm apart in each bed. Following the inoculation the nematodes were increased on cucumber (Cucumis sativus cv. Cobra) during spring 2013, and BHN 602 in the fall The trial was arranged in a randomized split-plot design with five treatments and six replications; five root-knot nematode species comprised the main-plots, and cultivar types: Crista and BHN 602 comprised the sub-plots. For the spring 2014 and 2015 plant growth ratings, tomato harvest, root galling, nematode population densities, and soil temperature measurements were assessed in late June and late November for fall 2014, following the same methods as previously mentioned. All data were subjected to analysis of variance (ANOVA) for split-plot design and mean were separated according to Tukey s honest significant difference (HSD) (SAS Institute, Cary, NC). Unless otherwise stated all differences listed in the results are at the P 0.05 level. Results Field Performance of Root-knot Resistant Tomato Cultivars Spring 2012 (Experiment one) The lowest plant growth ratings before final harvest were for Amelia nontreated and treated with 1,3-D (Table 2-1). The highest values where for BHN 602 and Amelia treated with 1,3-D + Pic. BHN 602 nontreated and treated with 1,3-D had similar intermediate values. 51

52 The yield of extra-large and large fruit was consistently higher for BHN 602 than for Amelia in plots treated with 1,3-D + Pic, whereas medium size fruit were similar between both cultivars in plots treated with 1,3-D + Pic. Yields for the 1,3-D treatment for both cultivars were intermediate between nontreated control and 1,3-D + Pic. There were no differences among yields between the two cultivars for the three fruit sizes or the total marketable yield for the nontreated controls. Both cultivars averaged 64 to 65% less when nontreated than the highest yield of BHN 602 treated with 1,3-D + Pic. The highest marketable yields were obtained from the treatment 1,3-D + Pic for both cultivars BHN 602 and Amelia, nevertheless, Amelia yielded 12% less than BHN 602. Again, the 1,3-D treatment yield was intermediate between the nontreated and 1,3- D + Pic treatment. The galling percentages ranged from 0 to 39% with the highest mean recorded for the nontreated BHN 602. All plots treated with fumigants resulted in negligible rootknot nematode galling and Amelia also had very low gall ratings in all plots. Spring 2012 (Experiment two) The highest values for plant growth responses taken before final harvest were greater for all three cultivars when treated with 1,3-D + Pic as compared with the nontreated control (Table 2-2). The growth rating for Red Bounty treated with 1,3-D + Pic was less than that for BHN 602 but not different from that of Amelia. Yields of extra-large, large and medium fruit were consistently higher than those of the nontreated control for all three cultivars when treated with 1,3-D + Pic. The marketable yield for BHN 602 treated with 1,3-D + Pic was higher than that for Amelia or Red Bounty. Both yielded ca. 17% less when compared with the highest value for BHN 602. Both Amelia and Red Bounty had similar values for marketable yield as compared 52

53 with the nontreated controls. The nontreated marketable yields ranged from 60 to 64% less than the highest yield value obtained for BHN 602 treated with 1,3-D + Pic. Both Amelia and Red Bounty, and all plots treated with1,3-d + Pic had less than 1% root galling, whereas the nontreated BHN 602 had a mean gall percentage of 58%. Fumigant Effects on Root-knot Nematode Resistance in Tomato Spring, Fall 2013, Spring 2014 For all three trials, there were no interactions between tomato cultivars and treatment variables; thus the comparisons were made only within treatments or within cultivars (Table 2-3, 2-5 and 2-7). However, for galling percentage there were significant interactions and the data was analyzed separately for each cultivar (Table 2-4, 2-6 and 2-8). Spring 2013 Plant growth vigor was greater in plots treated with metam K compared with the nontreated control and 1,3-D + Pic. Both tomato cultivars had similar growth responses (Table 2-3). Pic and MBr were the only treatments that produced higher marketable yields than the nontreated control. Yields in all other treatments were similar. Compared with Pic and MBr all other treatments resulted in 17 to 32% less yield. Crista produced 14% less marketable yield than BHN 602. The percentages for root-knot nematode galling on BHN 602 among the different treatments range from 0-51%, with the nontreated control, metam K, and Pic treatments having the highest percentages (Table 2-4). Mbr, 1,3-D + Pic, and 1,3-D all reduced percent galling compared with the nontreated control and metam K. Between cultivars, 53

54 BHN 602 had a higher percent galling in the nontreated control when compared with the resistant tomato cultivar. When analyzed separately the galling percentages among treatments for the two cultivars, Crista had values that ranged from 0-10%, whereas BHN 602 had values that ranged from 0-51% (Table 2-4). For BHN 602, the nontreated control, metam K, and Pic treatments had higher galling than the other four treatments, however Pic was not different from the nontreated control or metam K treatment. BHN 602 treated with Mbr, 1,3-D + Pic and 1,3-D had less galling than the nontreated control (0, 12, and 14%, respectively). On the other hand, galling on Crista was10% or less for all treatments including the nontreated control (Table 2-4). For spring 2013, temperatures and heat units were recorded from April 1 to June 18 (Figures 2-1, 2-2). At the beginning of the growing season (April) daily average temperatures above 28 C were recorded only for the 23-cm depth April 11, 17 and 20; however, from May 27 to 30 and June 8 to 18 all depths had average temperatures 28 C (Figure 2-1). Daily heat units were always higher for the 8-cm depth reaching the maximum of 23 hours on June 13 and 14. The maximum heat units for 15 and 23 cm depths were ca.13 hours at the end of June (Figure 2-2). Fall 2013 The best plant vigor ratings were for metam K, DMDS, Pic and 1,3-D + Pic (Table 2-5). The lowest ratings were for the nontreated control, and 1,3-D. Crista had a lower growth response than BHN 602. Yields of extra-large and large fruit were higher for metam K, Pic, DMDS, and 1,3-D + Pic than the nontreated control. There were no differences between nontreated control and 1,3-D. Between cultivars, BHN 602 had higher yield for all fruit sizes including marketable yields. Yield suppressions ranged 54

55 from 24% (DMDS) to 86% (nontreated control). Yields of Crista were 38% lower than those of BHN 602. Crista had a very low amount of galling regardless of treatments compared with BHN 602. All fumigants treatments except metam K and Pic reduced galling compared with the nontreated control (Table 2-6). For fall 2013, temperatures and heat units were recorded from August 23 to October 31 (Figures 2-3, 2-4). For all depths temperatures 28 C were recorded from August 23 to September 23 and October 3 to 7; and the lowest temperature 21 C was recorded at the end of growing season October 24 to 28 (Figure 2-3). Daily heat units were high at the 8, 15 and 23 cm depths reached the maximum of 23 hours from August 27 to September 23 (Figure 2-4). Spring 2014 The plant vigor ratings were higher for all treatments compared with the nontreated control (Table 2-7). Both Crista and BHN 602 had similar plant vigor responses. Yield of all fruit sizes were higher for all treatments except 1,3-D compared to the control. Between cultivars BHN 602 yielded more extra-large fruit when compared with Crista. For marketable yield, all treatments except 1,3-D were higher compared with the nontreated control. Yields were suppressed up to 39% for the nontreated control compared with the highest yield in the metam K treatment. Between cultivars BHN 602 had a 15% higher marketable yield than Crista. Percent galling on BHN 602 was reduced by all treatments. Among all treatments, Crista had very low galling (Table 2-8). For spring 2014, temperatures and heat units were recorded from March 25 to June 18 (Figures 2-5, 2-6). During the growing season, for all depths, there were five high temperature peaks ranging from 28.5 to 30 C. The highest average temperature 55

56 reached was 30 C on June 6 and the longest period with consecutive average temperatures above 28 C was between May 6 and 15 (Figure 2-5). The daily heat units were higher than in fall 2013 for 15 and 23 cm depths, particularly in the middle of May and June with almost 23 hours accumulated (Figure 2-6). Effects of Different Root-Knot Nematode Species on Resistant Tomato Spring 2014 There was no interaction between tomato cultivars and RKN species for variables tested, thus the comparisons were made only within RKN species or within cultivars (Table 2-9). However, for galling percentage there were significant interactions and the data was analyzed separately for each cultivar (Table 2-10). The plant growth responses were low for plots infested with all five root-knot nematode species. Between cultivars Crista had slightly higher plant growth response values when compared with BHN 602 (Table 2-9).Tomato fruit size extra-large, and total marketable yield were similar among plots infested with M. incognita, M. arenaria, M. floridensis, and M. enterolobii when compared with M. javanica (Table 2-9). When comparing the two cultivars, Crista had higher yields than BHN 602. The total marketable yield suppression for BHN 602 was 35%.Yields of large, and medium tomato fruit sizes were higher for M.incognita, and lower for M. javanica. When comparing cultivars BHN 602 had higher yield for tomato fruit size large, and lower yield for fruit size medium, when compared with Crista. On BHN 602 galling percentages were higher among all plots infested with rootknot nematode species, whereas on Crista there was 14 or less percentage of galling on roots parasitized by M. incognita, M. javanica, and M. arenaria (Table 2-10). 56

57 However, M. floridensis and M. enterolobii produced galling on Crista of 42 and 78%, respectively. Fall 2014 There was no interaction between tomato cultivars and RKN species for fruit sizes or total marketable yield, thus the comparisons were made only within RKN species or within cultivars (Table 2-11). However, for galling percentages there was interactions and the nematode effect on each cultivar was analyzed separately (Table 2-12). Plant vigor was higher for M. incognita and M. javanica. Between cultivars the best rating for plant vigor was for BHN 602 when compared with Crista (Table 2-11). Extra-large fruit size and marketable yield were higher for plots infested with all rootknot nematode species with the exception of M. enterolobii where the yield was low. Both cultivars had similar yields. Both cultivars had similar yield, however for marketable yield Crista had a yield decrease of 13% when compared with BHN 602. Tomato fruit sizes large and medium were similar among plots infested with all root-knot nematode species. Between cultivars, BHN 602 had significant higher large tomato fruit size yield when compared with Crista, but for medium tomato fruit size there were no differences between cultivars. For Crista, both M. enterolobii and M. floridensis induced significant galling, whereas 16% or less galling was induced on Crista by the other three root-knot nematode species. On BHN 602 galling was higher among all plots infested with rootknot nematode species (Table 2-12). For fall 2014, temperatures and heat units were recorded from August 25 to November 4 (Figures 2-7, 2-8). For all depths, high temperatures were recorded from 57

58 August 25 to the end of September. The maximum average temperature was 32 C reached August 31 (Figure 2-7). Daily heat units were higher at 15 and 23 cm depths at the beginning of the growing season (23 hours) and at the end of the growing season at the 7-cm depth where the highest values were recorded (4 hours) (Figure 2-8). Spring 2015 There was no interaction between tomato cultivars and root-knot nematode species thus the comparisons were made only within root-knot nematode species or within cultivars (Table 2-13). However, for galling percentages there was an interaction and the data was analyzed separately for each cultivar (Table 2-14). The plant vigor values were similar among all root-knot nematode species. However, between cultivars there were differences, with Crista having a slightly higher value when compared with BHN 602 (Table 2-13). For tomato fruit size extra-large, plots infested with M. incognita, M. javanica, and M. arenaria had higher yields when compared with plots infested with M. enterolobii. Tomato fruit size large and medium had lower yields for M. enterolobii, when compared with plots infested with M. arenaria. On marketable yield, all plots infested with root-knot nematodes with the exception of plots infested with M. enterolobii had similar yields. Between cultivars yields were similar for tomato fruit sizes extra-large, large, and marketable, whereas for tomato fruit size medium BHN 602 had lower yield when compared with Crista. Although, Crista and BHN 602 had similar marketable yields, BHN 602 had an apparent yield suppression of 12%. On BHN 602 relative gall ratings for all five root-knot nematode species were high. M. enterolobii, and M. javanica had higher galling percentages than plots infested 58

59 with M. incognita. Meloidogyne arenaria and M. floridensis had galling intermediate of that of M. enterolobii and M. javanica (Table 2-14). For Crista, the lower values of marketable yield were for plots infested with M. enterolobii. All others were similar including that for the DMDS treatment. For galling, both M. enterolobii and M. floridensis had high galling, whereas M. incognita, M. javanica, and M. arenaria had relatively low amount of galling. For spring 2015, temperatures and heat units were recorded from March 26 to June 12 (Figures 2-9, 2-10). During the growing season, for all depths, there was six high temperature peaks, ranging from 29 to 30.5 C. The highest average temperature reached was 30.5 C on April 6 and May 9 and 25. Daily heat units were consistently high after May 4 for all depths and reached the maximum of ca. 23 hours, 11 times for 15 and 23 cm depths (Figure 2-10). Discussion Spring seasons tended to have higher soil temperatures (above 28 C) at the middle and end of the growing season, whereas fall seasons tended to have higher temperatures at the beginning of growing season. The average soil temperature at 8, 15 and 23 cm-deep, tend not to differ too much, whereas for heat units (number of minutes per day recorded above 28 C) the values vary. From all soil temperature records, the maximum daily average ranged from 30 to 32 C and the maximum daily heat units accumulated were 1,400 minutes (approximately 23 hours). Based on root galling percentages, there was no evidence of a nonfunctioning of the Mi-gene under any of the different temperatures. Independently of the daily average temperatures and heat units, the values of root galling for RKN resistant tomato cultivars were lower than for RKN susceptible tomato cultivars regardeless of fumigant 59

60 treatment or soil temperature. The values ranged from zero to 10%, indicating that resistant tomato cultivars are not immune to RKN and a few juveniles were able to infect roots, but they develop slowly resulting in a reproduction rate smaller than in a susceptible tomato cultivar. Similar results have been reported under field conditions on alfalfa (Griffin and Elgin, 1977), soybean (Pedrosa et al., 1996) and tomato (Roberts et al., 1986; Olson and Rich, 1999). The root galling percentages in these experiments are in agreement with research conducted by Olson and Rich (1999), and Roberts et al. (1986). Independently of the treatment used RKN resistant tomato cultivars had low root galling (0 to 10%), whereas RKN susceptible tomato cultivars had low root galling when treated with an effective soil fumigant (0 to 14%). There have been few studies done evaluating RKN resistant tomato cultivars under field conditions (Roberts et al., 1986; Olson and Rich, 1999). In North Florida, Olson and Rich (1999) conducted two field trials (fall 1997 and spring 1998) and compared root galling percentages, and fruit weight and number of two RKN susceptible tomato cultivars (Agriset 761 and Colonial cvs.) and two RKN resistant tomato cultivars (Sanibel and PSR cvs.) in a field inoculated with M. javanica. Their results suggested that RKN resistant tomato cultivars are not well adapted to Florida field conditions since yields of Agriset 761 and Colonial were higher than those of Sanibel and PSR These results are in agreement with the results from this research. For all our field trials there was an estimated yield loss between 10 to 38% between the RKN resistant tomato cultivar and susceptible tomato cultivar when root-knot nematodes 60

61 were low or a non-factor. The lower yields of 10 and 38% were for Crista where there was an evaluation of fumigant effects on root-knot nematode resistant tomato, on spring and fall 2013, respectively. However, these results are not in agreement with an experiment conducted in California by Roberts et al. (1986). They conducted three field trials comparing yields and root galling percentages among RKN resistant tomato cultivars and lines, and treatments (nontreated and 1,3-D). In their trials there were no differences between treated and nontreated RKN resistant tomato cultivars, there were differences between treated and nontreated susceptible tomato cultivars. Roberts et al., (1986) concluded that the ability of resistant tomatoes to produce yields in a RKN infested field was so promising that they could be grown safely without additional nematode management inputs such as nematicides. However, in trials reported here yields of both root-knot nematode resistant and susceptible cultivars were different between 1,3-D + Pic and nontreated in experiments one and two of the field performance of root-knot resistant tomato cultivars. This indicates that under Florida conditions RKN resistant tomato may still benefit from soil fumigation. Further, while our results confirm that Mi-gene resistance was effective in reducing damage from Meloidogyne spp., when planted without fumigation yields would be suppressed in most instances. Whether this is a reflection of the germplasm or not it is unknown. The cultivars used in these studies are recommended for planting in Florida (Bloem and Mizell, 2004). The fumigants included in this research that provided greatest benefits were MBr (spring 2013), and metam K (fall 2013 and spring 2014). Yields for 1,3-D treated (fall 61

62 2013, and spring 2014) were not different from the nontreated. It is unclear why 1,3-D performed so poorly in the field infested with root-knot nematodes, maybe it was related with the low dosages applied. However, other fumigants with nematicidal activity namely Mbr and DMDS performed very well. On some trials metam K also perform well in terms of marketable yields. The possible reason of higher yields for metam K might have been related with the extra source of potassium when compared with other fumigants, and also related with the management of soilborne fungal pathogens and weeds, although no discernable soilborne fungal agents were identified. The importance of using resistant tomato cultivars in combination with broadspectrum fumigants also shows the potential inefficiency of resistance in the presence of some RKN species like M. enterolobii and M. floridensis. These two species were reported to break resistant genes in several crops including tomato (Brito et al., 2004, 2007a, 2007b; Church, 2005). In our study based on root galling percentages, it was clear that Mi-gene resistance was nonfunctional in the presence of both species. However, the differences in virulence between M. enterolobii and M. floridensis was consistent in that M. enterolobii was noted as a more virulent pathogen. Several factors could have led to this results including population density values in the soil, resistance mechanisms in tomato,and differences among M. floridensis populations. This particular isolate came from a peach site in Gainesville, Florida, and consequently the isolate may be less virulent in tomato. The Mi-gene resistance has a constant low yield performance when compared with RKN susceptible tomato cultivars. Several hypotheses can be related with this fact. It can be related with costs of resistance expression when in the presence of a 62

63 pathogen (induced resistance). Or it can be net costs where resistant plants may have lower fitness than susceptible plants when soilborne pathogens are present; or allocation cost where resistance expression reduces sources available for growth and reproduction; or ecological costs where resistance expression reduces plant s ability to resist or tolerate other pests or pathogens. It was reported by Beynon (1997) that R- genes are prone to net costs and susceptibility may be favored over resistance if a pathogen is weak or if the environmental conditions do not favor the development of severe disease symptoms. Few studies have been conducted to analyze the effects of R-genes on plant reproductive success, Brandon et al., 2011 conducted an experiment to assess the effects of nematode infection and Mi-mediated resistance on plant growth and reproduction with the goal to evaluate the potential fitness costs and benefits of Mi-gene and to explore the potential role of nematodes as a selection pressure favoring Mimediated resistance. He concluded that susceptible tomato cultivar utilize tolerance mechanisms to compensate for moderate levels of nematode infection, whereas the Mi- 1,2 gene resistance confered a dramatic fitness benefit under heavy nematode pressure. The physiology and molecular mechanism underlying tolerance are not well understood, but are known to involve realocation of resources such as photoassimilates to less vulnerable parts of the plant (Agrawal et al., 1999). Nicotiana attenuate relocate sugars to roots in response to simulated herbivore attack, for storage and future regrowth (Schwachtje et al., 2006). Understanding the real cost of resistance and 63

64 tolerance in the tomato cultivars planted in varying soils and environmental conditions are needed. One of the potential limitations in using root-knot nematode resistant tomato cultivars to manage root knot disease in Florida is related with soil temperature. It has been reported by several investigators that there was a reduction of the expression of the Mi-gene at temperatures above 28 C (Holtzman, 1965; Dropkin, 1969; Araujo et al., 1982a, b; Ammati et al., 1986; Haroon et al., 1993; Wang et al., 2009; Devran et al., 2010). When analyzing the soil temperature measurements of this study it was clear that in both fall and spring seasons from 2013 to 2015, and independently of the temperatures at different soil depths, there were records of daily mean temperatures that would be projected to lead to the nonfunctioning of the Mi-gene. The calculated daily heat units had periods of time of almost 24 hours where the temperatures were above 28 C for almost 1, 20 and 23 days for fall 2013 and 2014, respectively. In general, soil temperatures were frequently above the threshold of 28 C previously, reported to cause nonfunctioning of the Mi-gene. Dropkin (1969) reported that resistance conferred by the Mi-gene was not effective when plants were inoculated and incubated at 32 C for three consecutive days followed by incubation at 27 C, but effective when plants were inoculated and incubated at 27 C for 2 days and then 32 C. Therefore, it is possible that the impact of temperature on Mi-gene would be greater in fall than in spring growing season. The fall season had higher temperatures in the beginning of the growing season when tomatos were transplanted and were more vulnerable to infection by different RKN species. 64

65 Tzortzakakis et al. (2000) reported that resistance in tomato was not overcome in a March- through August crop in a plastic house in Crete because soil temperatures above 28 C were registered throughout June- through August. In our study 17 days with soil temperatures above 28 C in the last month of the crop cycle were insufficient to suppress the resistance response in the resistant cultivar. In general, Mi- gene by itself is not an alternative to chemical management in Florida due to the consistent yield suppressions of RKN resistant tomato cultivar when compared with RKN susceptible tomato cultivars; however it remains as a separate tactic in an IPM program, particularly for the management of M. floridensis and M. enterolobii. More research is necessary particularly comparing resistant tomato cultivars available with near-isogenic lines. This evaluation would give us a possible answer about the yield suppression since with an introduction of a resistant gene there is always extra unwanted DNA accompanying. 65

66 Table 2-1. Effect of soil fumigant treatments and tomato cultivars BHN 602 (susceptible) and Amelia (resistant) on plant vigor, yield and root-knot nematode galling in a field trial on spring 2012 (Experiment 1). Treatment Rating 1 XL L M (rate/ha) (0-10) (kg/ha) (kg/ha) (kg/ha) Total marketable Galling yield 2 percentages 3 (kg/ha) BHN 602 Nontreated 4.7 b 1,838 d 3,063 de 5,839 c 10,740 c (65) a 1,3-dichloropropene + chloropicrin 325 liters 9.5 a 5,550 a 8,974 a 16,146 a 30,670 a (0) 0.4 b 1,3-dichloropropene 140 liters 5.4 b 3,676 bc 5,262 c 8,073 b 17,011 b (45) 2.1 b Nontreated 3.3 c 1,910 d 2,811 e 6,163 bc 10,884 c (64) 0.2 b Amelia 1,3-dichloropropene + chloropicrin 325 liters 8.3 a 3,892 b 6,740 b 16,363 a 26,995 a (12) 0 b 1,3-dichloropropene 140 liters 3.8 c 2,487 cd 4,325 cd 7,641 bc 14,453 bc (53) 0 b Data are means of six replications in a complete block design. Means within a column followed by a common letter are not different according to Duncan s multiple-range test (P 0.05). 1 Rating at final harvest. Subjective plant vigor rating scale = 1 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Total marketable yield = sum of extra-large, large and medium tomato weights. 3 Subjective root-knot nematode galling based on the following scale: 0 100, where 0 = no galls on the root system; 10 = 10% 100 = 100% of root system galled. 4 Percentages of yield suppression compared with the highest yield value. 66

67 Table 2-2. Effect of soil fumigant treatment and tomato cultivars BHN 602 (susceptible), Amelia (resistant), and Red Bounty (resistant) on plant vigor, yield, and root-knot nematode galling in a field trial on spring 2012 (Experiment 2). Galling Treatment Rating 1 XL L M Total marketable yield 2 percentages (rate/ha) (0-10) (kg/ha) (kg/ha) (kg/ha) (kg/ha) 3 BHN 602 Nontreated 4.8 c 4,001 bc 4,433 c 6,018 b 14,452 c (60) 4 58 a 1,3-dichloropropene + chloropicrin 325 liters 9.7 a 8,506 a 12,974 a 14,632 a 36,112 a (0) 1 b Nontreated 4.2 c 3,027 c 3,892 c 6,199 b 13,118 c (64) 1 b Amelia 1,3-dichloropropene + chloropicrin 325 liters 8.6 ab 5,839 b 9,371 b 14,777 a 29,987 b (17) 0 b Red Bounty Nontreated 4.8 c 4,505 bc 3,640 c 6,199 b 14,344 c (60) 0 b 1,3-dichloropropene + chloropicrin 325 liters 8.4 b 9,118 a 8,001 b 13,154 a 30,273 b (16) 0 b Data are means of six replications in a complete block design. Means within a column followed by a common letter are not different according to Duncan s multiple-range test (P 0.05). 1 Rating at final harvest. Subjective plant vigor rating scale = 1 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Total marketable yield = sum of extra-large, large and medium tomato weights. 3 Subjective root-knot nematode gall index scale = 0 100, where 0 = no galls on the root system; 10 = 10% 100 = 100% of root system galled. 4 Percentages of yield suppression compared with the highest yield value. 67

68 Table 2-3. Effect of fumigant treatments and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on spring Rating 1 XL L M Total marketable yield 2 (0-10) (kg/ha) (kg/ha) (kg/ha) (kg/ha) Treatments (rate/ha) Nontreated 7.5 b 4,179 cd 3,662 c 3,174 b 11,015 b (32) 3 Metam potassium 561 liters 8.9 a 2,900 d 4,785 ab 5,224 a 12,909 b (20) Chloropicrin 263 kilograms 8.6 ab 6,162 a 5,322 a 4,619 a 16,103 a (1) Methyl bromide 393 kilograms 8.6 ab 5,586 ab 5,527 a 5,039 a 16,152 a (0) 1,3-dichloropropene 140 liters 8.3 ab 4,843 bc 3,916 bc 3,203 b 11,962 b (26) 1,3-dichloropropene + chloropicrin 325 liters 7.0 b 3,467 d 4,785 ab 5,146 a 13,398 b (17) Tomato cultivars BHN a 5,259 a 4,772 a 4,267 a 14,298 a (0) Crista 7.7 a 3,788 b 4,561 a 4,536 a 12,885 b (14) Data are means of five replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Ratings at final harvest. Subjective plant vigor rating scale = 1 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Total marketable yield = sum of extra-large, large and medium tomato weights. 3 Percentages of yield suppression compared with the highest yield value. 68

69 Table 2-4. Effect of fumigant treatment and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on spring Treatments (rate/ha) Galling percentages 1 BHN 602 Crista Nontreated 51 a 10 a Metam potassium 561 liters 39 a 6 a Chloropicrin 263 kilograms 26 ab 3 a Methyl bromide 393 kilograms 0 b 0 a 1,3-dichloropropene 140 liters 14 b 1 a 1,3-dichloropropene + chloropicrin 325 liters 12 b 0 a Data are means of five replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Subjective root-knot nematode gall index scale = 0 100, where 0 = no galls on the root system; 10 = 10% 100 = 100% of root system galled. 69

70 Table 2-5. Effect of fumigant treatments and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on fall Rating 1 XL L M Total marketable yield 2 (0-10) (kg/ha) (kg/ha) (kg/ha) (kg/ha) Treatments (rate/ha) Nontreated 5.0 c 25 b 83 c 340 d 448 d (86) 3 Metam potassium 561 liters 7.5 a 400 a 934 a 1,807 a 3,141 a (0) Chloropicrin 263 kilograms 6.8 ab 273 a 437 b 1,118 bc 1,828 bc (42) Dimethyl disulfide 374 liters 7.5 a 227 ab 632 ab 1,528 ab 2,387 ab (24) 1,3-dichloropropene 140 liters 5.5 bc 174 ab 420 bc 723 cd 1,317 cd (58) 1,3-dichloropropene + chloropicrin 325 liters 6.9 ab 283 a 642 b 1,263 b 2,188 bc (31) 70 Tomato cultivars BHN a 377 a 723 a 1,221 a 2,321 a (0) Crista 6.0 b 81 b 322 b 1,045 b 1,448 b (38) Data are means of five replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Rating at final harvest. Subjective plant vigor rating scale = 1 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Total marketable yield = sum of extra-large, large and medium tomato weights. 3 Percentages of yield suppression compared with the highest yield value.

71 Table 2-6. Effect of fumigant treatment and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on fall Treatments (rate/ha) Galling percentages 1 BHN 602 Crista Nontreated 71 a 5 a Metam potassium 561 liters 34 b 3 a Chloropicrin 263 kilograms 17 bc 2 a Dimethyl disulfide 374 liters 3 c 1 a 1,3-dichloropropene 140 liters 5 c 0 a 1,3-dichloropropene + chloropicrin c 2 a liters Data are means of five replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Subjective root-knot nematode galling based on the following scale: 0 100, where 0 = no galls on the root system; 10 = 10% 100 = 100% of root system galled. 71

72 Table 2-7. Effect of fumigant treatments and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on spring Rating 1 XL L M Total marketable yield 2 (0-10) (kg/ha) (kg/ha) (kg/ha) (kg/ha) Treatments (rate/ha) Nontreated Metam potassium 561 liters Chloropicrin 263 Kilograms Dimethyl disulfide 374 liters 1,3-dichloropropene 140 liters 1,3-dichloropropene + chloropicrin 325 liters 5.2 b 4,795 b 2,871 c 2,305 c 9,971 d (39) a 6,543 ab 5,420 a 4,355 a 16,380 a (0) 7.6 a 5,703 ab 4,026 b 3,447 b 13,176 bc (19) 7.6 a 6,836 a 4,521 b 3,525 b 14,842 ab (9) 7.4 a 6,191 ab 3,125 c 2,373 c 11,689 cd (28) 7.8 a 4,765 b 4,765 ab 4,234 ab 13,764 abc (16) Tomato cultivars BHN 602 Crista 7.3 a 7,031 a 4,199 a 3,174 a 14,404 a (0) 7.1 a 4,590 b 4,101 a 3,574 a 12,265 b (15) Data are means of five replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Rating at final harvest. Subjective plant vigor rating scale = 1 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Total marketable yield = sum of extra-large, large and medium tomato weights. 3 Percentages of yield suppression compared with the highest yield value. 72

73 Table 2-8. Effect of fumigant treatments and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on spring Treatments (rate/ha) Galling percentages 1 BHN 602 Crista Nontreated 27 a 1 a Metam potassium 561 liters 21 b 0 a Chloropicrin 263 kilograms 19 b 0 a Dimethyl disulfide 374 liters 10 c 0 a 1,3-dichloropropene 140 liters 11 c 0 a 1,3-dichloropropene + chloropicrin 325 liters 10 c 0 a Data are means of five replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Subjective root-knot nematode galling based on the following scale: 0 100, where 0 = no galls on the root system; 10 = 10% 100 = 100% of root system galled. 73

74 Table 2-9. Effect of root-knot nematode species, and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on spring Rating 1 XL L M Total marketable yield 2 (0-10) (kg/ha) (kg/ha) (kg/ha) (kg/ha) Treatments Meloidogyne incognita 3.3 a 206 a 195 a 337 ab 739 a (4) 3 Meloidogyne javanica 3.4 a 66 b 103 b 216 b 384 b (50) Meloidogyne arenaria 3.2 a 188 a 184 ab 394 a 766 a (0) Meloidogyne floridensis 3.0 a 157 a 157 ab 326 ab 640 a (16) Meloidogyne enterolobii 3.3 a 197 a 140 ab 306 ab 643 a (16) Tomato cultivars BHN b 96 b 174 a 267 b 537 b (35) Crista 3.7 a 230 a 138 b 365 a 733 a (0) Data are means of six replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Rating at final harvest. Subjective plant vigor rating scale = 1 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Total marketable yield = sum of extra-large, large and medium tomato weights. 3 Percentages of yield suppression compared with the highest yield value. 74

75 Table Effect of root-knot nematode species, and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on spring Galling percentages 1 BHN 602 Crista Meloidogyne incognita 94 a 6 b Meloidogyne javanica 98 a 9 b Meloidogyne arenaria 93 a 14 b Meloidogyne floridensis 84 a 42 b Meloidogyne enterolobii 97 a 78 a Data are means of six replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Subjective root-knot nematode galling based on the following scale: 0 100, where 0 = no galls on the root system; 10 = 10% 100 = 100% of root system galled 75

76 Table Effect of root-knot nematode species, and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield in a field trial on fall Rating 1 XL L M Total marketable yield 2 (0-10) (kg/ha) (kg/ha) (kg/ha) (kg/ha) Treatments Meloidogyne incognita Meloidogyne javanica Meloidogyne arenaria Meloidogyne floridensis Meloidogyne enterolobii 6.2 a 561 ab 685 a 1,474 a 2,720 ab (13) ab 662 a 797 a 1,451 a 2,916 ab (7) 5.8 b 463 ab 1,352 a 1,317 a 3,132 a (0) 6.8 b 531 ab 598 a 1,409 a 2,539 ab (19) 5.6 b 345 b 460 a 1,209 a 2,014 b (36) Cultivars BHN 602 Crista 6.6 a 501 a 977 a 1,365 a 2,843 a (0) 5.5 b 523 a 580 b 1,380 a 2,483 a (13) Data are means of six replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Rating at final harvest. Subjective plant vigor rating scale = 1 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Total marketable yield = sum of extra-large, large and medium tomato weights. 3 Percentages of yield suppression compared with the highest yield value. 76

77 Table Effect of root-knot nematode species, and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on fall Galling percentages 1 BHN 602 Crista Meloidogyne incognita 95 a 7 b Meloidogyne javanica 98 a 10 b Meloidogyne arenaria 93 a 16 b Meloidogyne floridensis 89 a 32 a Meloidogyne enterolobii 97 a 67 a Data are means of six replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Subjective root-knot nematode galling based on the following scale: 0 100, where 0 = no galls on the root system; 10 = 10% 100 = 100% of root system galled 77

78 Table Effect of root-knot nematode species, and tomato cultivars BHN 602 (susceptible) and Crista (resistant) on plant vigor, and yield s in a field trial on spring Rating 1 XL L M Total marketable yield 2 (0-10) (kg/ha) (kg/ha) (kg/ha) (kg/ha) Treatments Meloidogyne incognita Meloidogyne javanica Meloidogyne arenaria Meloidogyne floridensis Meloidogyne enterolobii 6 a 1,184 a 755 ab 1,154 ab 3,092 a (7) 3 6 a 1,139 a 692 ab 1,120 ab 2,951 a (11) 6 a 1,030 a 959 a 1,323 a 3,312 a (0) 6 a 806 ab 747 ab 1,308 a 2,861 a (14) 6 a 577 b 524 b 857 b 1,958 b (41) Cultivars BHN 602 Crista 5.6 b 985 a 678 a 988 b 2,651 a (12) 6.2 a 909 a 792 a 1,317 a 3,018 a (0) Data are means of six replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Rating at final harvest. Subjective plant vigor rating scale = 1 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Total marketable yield = sum of extra-large, large and medium tomato weights. 3 Percentages of yield suppression compared with the highest yield value. 78

79 Table Effect of root-knot nematode species, and the tomato cultivars BHN 602 (susceptible) and Crista (resistant) on root-knot nematode galling in a field trial on spring Galling percentage 1 BHN 602 Crista Meloidogyne incognita 68 c 9 d Meloidogyne javanica 87 ab 23 c Meloidogyne arenaria 81 bc 10 d Meloidogyne floridensis 71 bc 49 b Meloidogyne enterolobii 99 a 94 a Data are means of six replications in a split-plot design. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). 1 Subjective root-knot nematode galling based on the following scale: 0 100, where 0 = no galls on the root system; 10 = 10% 100 = 100% of root system galled 79

80 ºC cm 15 cm 23 cm April May June Figure 2-1. Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for spring

81 Minutes cm 15 cm 23 cm April May June Figure 2-2. Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for spring

82 C cm 15 cm 23 cm August September October Figure 2-3. Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for fall

83 Minutes cm 15 cm 23 cm August September October Figure 2-4. Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for fall

84 C cm 15 cm 23 cm March April May June Figure 2-5. Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for spring

85 Minutes cm 15 cm 23 cm March April May June Figure 2-6. Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for spring

86 C cm 15 cm 23 cm August September October November Figure 2-7. Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for fall

87 Minutes cm 15 cm 23 cm August September October November Figure 2-8. Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for fall

88 C cm 15 cm 23 cm March April May June Figure 2-9. Mean of daily soil temperatures at three different depths (8, 15, and 23 cm) for spring

89 Minutes cm 15 cm 23 cm March April May June Figure Daily heat units of soil temperature above 28 C for three different depths (8, 15, and 23 cm) for spring

90 CHAPTER 3 EVALUATION OF THE RESIDUAL EFFECT OF ROOT-KNOT NEMATODE RESISTANT TOMATO CULTIVARS AND FUMIGANTS IN A DOUBLE-CROP SYSTEM Introduction Multiple cropping has been practiced for centuries in many regions of the world, but in the United Sates the practice began during the mid s. By the 1970 s it became more mainstay mainly because of crop prices and agricultural innovations (Hexem and Boxley, 1986). In general, multiple cropping is more risky and costly than a single crop. Its success depends on numerous factors, e.g., weather (length of growing season, frosting and precipitation periods), logistics (machinery and labor force), and national and world economies. Proper crop management is crucial for the success of intensive crop production systems. Most multiple cropping in the United States is sequential double cropping and to a lesser extent, relay intercropping (two crops that grow simultaneously during part of the life-cycle of each). Sequential double cropping is the practice of one successive planting on existing polyethylene or organic mulched beds, meaning that two crops are grown in a period of 1 year (Gallaher, 1995). In Florida, various vegetables have been grown in a sequential double cropping system: muskmelon, honeydew, watermelon, squash, cucumber, tomato, pepper and eggplant. The practice was aided by the development of polyethylene films in the early 1950 s (Wright, 1968). The polyethylene films (PE) provided many positive advantages for the user, such as increased yields, earlier-maturing crops, higher-quality produce, insect management, weed control, and the prevention of excessive leaching of fertilizer. It also allows other components such as drip irrigation to achieve maximum efficiency. However, the use of PE is costly and specific machinery is required, therefore it is 90

91 important to maximize its usage in a sequential double cropping system (Lament, 1993). The Clean Water Act that regulates the quantity and quality of pollutants into the waters of the United States also played an important role on the fast implementation of a sequential crop system in PE covered beds. The double cropping system improves the efficacy of broad-spectrum fumigants thereby effectively extending periods of their use, and also utilizes unused (residual) fertilizers (Simonne and Hochmuth, 2003). Sequential double cropping also can be viewed as a cultural management tool for soilborne pests and pathogens, particularly root-knot nematodes (RKN). The practice can serve as a type of crop rotation where non-host crops (e.g. marigold), resistant crops (e.g. Mi-gene tomato), or grafted crops provide a basis for reducing RKN population densities in soil (Trivedi and Baker, 1986). In a sequential multiple crop system, the use of tomato with the Mi-gene followed by a susceptible crop has been studied (Hanna et al., 1993; Ornat et al., 2001; Rich and Olson, 2004; Thies et al., 2004). Some studies used different RKN resistant tomato cultivars followed by cucumber. These studies showed that the yields of a second crop were greater because of the reduction of RKN soil population densities on the first crop (Hanna et al., 1993; Ornat et al., 2001; Thies et al., 2004). Similar results were obtained using a resistant tomato-cantaloupe sequence (spring summer growing seasons) in north Florida (Rich and Olson, 2004). Cantaloupe yields were increased and galling percentages were lowered when using a combination of soil fumigants and root-knot resistant tomato cultivars. Since some portions of Florida provides climatic conditions conductive for the production of several crops per year in a sequential multiple cropping system, it is 91

92 important to continue to evaluate the effectiveness of using tomato cultivars with the Migene resistance to manage RKN populations in fall-winter and spring-summer growing seasons. Two studies were conducted to determine efficacy of soil fumigants, root-knot nematode resistant tomato, and the double cropping of tomato followed by cucumber - Cucumis sativus L. (spring summer season) or carrot - Daucus carota subsp. sativus L. (fall-winter seasons). Material and Methods The field trials were conducted in fall 2013 and spring 2014 in a sequential crop system that followed tomato. Carrot Trial In the fall 2013 trial hybrid carrot cv. Maverick (Siegers Seed Co., IL) followed tomato. Carrots were seeded in early December after removing the polyethylene film and rototilling the soil. Approximately one seed per centimeter was sowed in a single row at a depth of 0.6 centimeter in the bed middle were tomato was previously grown. The field design used previously for the tomato trial was a split-plot with five replicates with root-knot nematode resistant and susceptible tomato as main plots and five soil fumigants as subplots. The carrots were watered biweekly via a center pivot with drop sprinklers until cracking, followed by once weekly, if needed. One month after planting the plots were fertilized with (N-P 2 O 5 -K 2 O) at a rate of 447 kg/ha. One rating of plant vigor was done as the crop reached maturity in March. The rating was based in a subjective scale of one to 10; with 1 = small, chlorotic and nonvigorous plants and 10 = large, dark green and vigorous plant foliage. 92

93 Tap roots were harvested in early April and graded into two categories: marketable and nonmarketable. Marketable carrots were based on size that was equal or greater than 12-cm, and no root-knot nematode galling or forking of the primary root; and nonmarketable carrot was based on tap roots that were less than12-cm, and with significant galling and forking of the primary root (Figure 3-1). Root-knot nematode population densities in soil were assessed at the end of the tomato harvest and at carrot harvest. Six soil cores were taken to a depth of 20-cm in each subplot using a 2.5-cm-diam cone-shaped soil probe and combined. A 100-cm 3 subsample was used to extract nematodes using the centrifugal-flotation technique (Jenkins, 1964). The J2 were counted using an inverted microscope at x40 magnification. All data, except that mentioned below, were subjected to analysis of variance (ANOVA) for split-plot design and mean separations were determined by Tukey s honest significant difference (HSD) (SAS Institute, Cary, NC). A reproductive factor was determined dividing the J2 population density at carrot harvest by the initial population density at tomato harvest. This data and initial and final root-knot nematode population densities were compared using a Student s t-test. Unless otherwise stated all significant differences listed are at the P 0.05 level. Cucumber Trial Following the spring 2014 tomato planting cucumber cv. Cobra seedlings were transplanted as a sequential crop in the former tomato plant holes. Cucumbers were fertilized via the drip system once weekly with urea 42% and potassium chloride 62%, for a total of 168 kg/ha for N and 133 kg/ha for K 2 O applied over the crop season. 93

94 Foliar fungal pathogens were managed using alternating spray applications of chlorothalonil, mancozeb and azoxystrobin; and soilborne pests were managed by drip applied dinotefuran. Weeds in the row middles were managed with a mixture of metribuzin, napropamide, halosulfuron-methyl, metalochlor and trifluralin applied before transplanting. At final harvest vigor rating was done based on the scale mentioned previously for carrot. Cucumbers were harvested twice weekly for 4 weeks, from mid-october to mid-november and graded as marketable and nonmarketable. Marketable cucumbers were without malformation and 8 to 10 inches long. After the final harvest, 10 plant roots (chosen arbitrarily) per sub-plot were subjected to a root-gall rating based on a 0 to 100% scale where 0 = no visible galls, 10 = 10% of root system galled, and 100 = 100% of the root system galled (Barker et al., 1986). Nematode population densities in soil and data analysis followed the same procedure as mentioned previously for the carrot trial. Results For both carrot and cucumber there were no interactions between tomato cultivars and soil fumigants, thus the comparisons were made only within treatments or within cultivars (Tables 3-1, 3-3). However, between the root-knot nematode population densities in soil and reproductive factors there were interactions (Tables 3-2, 3-4). Carrot Fall 2013 Plant vigor results were similar among treatments but differed between cultivars. The vigor of carrot following plots planted with Crista were greater than those following BHN 602 (Table 3-1). 94

95 For DMDS and 1,3-D + Pic treatments the number of marketable carrots were higher than that in the nontreated control. Other treatments were not different. There were 12 to 44% fewer carrots among treatments compared with the highest carrot numbers in the DMDS treated plots. There were no differences in the number of carrots harvested between the two tomato cultivars although there was 17% less in Crista plots. There was an increase in marketable yields compared with the nontreated among all treatments except for metam K. There were no differences between the two tomato cultivars, although there was 16% less in Crista plots. Among all treatments there were no differences for nonmarketable numbers or weight, however, there were higher numbers numerically in plots previously planted with Crista. Cultivars and treatments had an impact on J2 soil population densities before transplanting carrots. The initial population densities before transplanting tomato ranged from 51 to 124 J2/100 cm 3 of soil for both cultivars (Table 3-2). Following soil fumigation and the tomato crop, final J2 population densities were lower in plots with Crista leading to a low reproductive factor and higher in plots with BHN 602 leading to a high reproductive factor; however, for DMDS treatment the reproductive factor was lower for both cultivars. Among treatments, the highest J2 population densities were for nontreated and Pic treatment for Crista and metam K for BHN 602. Also Pic had similar numbers to that in the nontreated control, but was less than in the metam K treatment. The lowest numbers of J2 were in the DMDS treatment for both Crista and BHN 602. Among fumigants, reproductive factors at the end of tomato growing season were low for Crista (ranging from 0.1 to 0.5) when compared with BHN 602 (ranging from 0.3 to 20). 95

96 When comparing J2 final population densities between tomato and carrot growing seasons there was an increase in plots previously planted with Crista, whereas for plots previously planted with BHN 602 there was a decrease except for DMDS and nontreated control. The same trend occurred for Rf, the values for BH602 ranged from 0 to 2.9, whereas for Crista they ranged from 2.2 to The highest Rf values were for metam K for Crista, and DMDS for BHN 602. Cucumber spring 2014 Plant vigor ratings between previous fumigant treatments and cultivars were similar ranging from 7.7 to 8.2 (Table 3-3). Only metam K and 1,3-D + Pic had higher marketable numbers and weight than the nontreated control. All other treatments were similar. When comparing root galling percentages, the previous plantings of Crista resulted in had lower galling values on cucumber compared with BHN 602 (Table 3-3). For both tomato cultivars, the initial populations were similar for both tomato cvs., however the final population was higher following BHN 602 compared to Crista among all treatments except Pic (Table 3-4). Following the plantings of cucumber, the J2 population densities were consistently lower in the former plots than in previous BHN 602 plots. Following cucumber there was higher number of J2 in the Pic treatment than in the metam K, Dmds, and 1,3-D + Pic treatments. The Rf values were lower for both tomato and cucumber crops following Crista. Discussion Double or sequential cropping is a common practice particularly as a way to increase returns after a high value crop. In Florida, this practice is common following tomato, pepper, or strawberry. Due to the ability of RKN resistant tomato to work as a suppressive/trap crop it continues to offer important possibility for Florida producers. In 96

97 Florida only one study has evaluated a double cropping systems following a planting of tomato cultivars containing Mi-1 genewhere tomato was the double-cropped with cantaloupe (Rich and Olson, 2004). We evaluated two double crop systems in to distinct growing seasons. In both, there was no benefit from using root-knot nematode resistant tomato cultivars other than reducing the J2 population densities in the soil. Similar results have been reported (Hanna et al., 1993; Thies et al., 2004; Ornat et al., 2010); however, in their studies there was an increase in the yield of second crop, whereas in these experiments no increase in yield was obtained with carrot or cucumber following the previous plantings of RKN resistant and susceptible tomato cultivars. There were residual effects of soil fumigant treatments. Both DMDS and metam K resulted in higher yields for carrot and cucumber. Oddly, there were no residual effects following treatments of 1,3-D or 1,3-D + Pic. Similar results were previously reported (Rich and Olson, 2004). Metam K treatment resulted in better cucumber yields, which might be related with the ability of metam K to manage soilborne fungi besides providing an extra source of potassium. In general, RKN resistant tomato supressed the RKN population but the previous treatment with multi-purpose soil fumigant DMDS was a stronger component in the system. 97

98 Figure 3-1. Photograph illustrating marketable vs. nonmarketable carrot. Nonmarketable carrots were forked, galled, or both symptoms. A) Marketable carrot; B) Nonmarketable carrot. Courtesy of author. 98

99 Table 3-1. Residual effect of fumigant treatments and tomato cultivars in a double crop system with carrot following tomato. Rating 1 Marketable 2 Marketable 2 Nonmarketable 3 Nonmarketable 3 (0-10) nu/ha kg/ha nu/ha kg/ha Treatments 4 (rate/ha) Nontreated 6 a 12,702 b (52) 937 b (46) 26,479 a 1,016 a Metam potassium 561 liters 7 a 14,747 ab (44) 1,133 b (35) 26,264 a 810 a Chloropicrin 263 kilograms 8 a 19,935 ab (25) 1,484 a (15) 22,820 a 771 a Dimethyl disulfide 374 liters 8 a 26,566 a (0) 1,738 a (0) 24,111 a 713 a 1,3-dichloropropene 140 liters 8 a 19,203 ab (28) 1,416 a (19) 21,528 a 723 a 1,3-dichloropropene + chloropicrin 325 liters 7 a 23,315 a (12) 1,709 a (2) 23,896 a 703 a Tomato cultivars BHN b 21,199 a (0) 1,523 a (0) 20,630 b 734 a Crista 8 a 17,625 a (17) 1,279 a (16) 27,679 a 843 a Split-plot design with five replicates. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). Parenthesis indicates percentage yield loss compared with the highest yield value. 1 Subjective plant vigor rating scale = 1 to 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Marketable carrot based on 12-cm in length, and no galling or forking of the tap root. 3 Nonmarketable carrot based < 12-cm in length, and galling and forking visible on the tap root. 4 Rate is broadcast equivalent. 99

100 Table 3-2. Residual effect of fumigant treatments and cultivars BHN 602 (susceptible) and Crista (resistant) on secondstage juvenile population densities in soil and the reproductive factor in a double crop system with carrot following tomato. Tomato Carrot Treatments (rate/ha) 1 Initial second-stage juvenile nu/100 cm 3 of soil Final second-stage juvenile nu/100 cm 3 of soil Rf 2 Final second-stage juvenile nu/100 cm 3 of soil Rf 2 Crista BHN 602 Crista BHN 602 Crista BHN 602 Crista BHN 602 Crista BHN 602 Nontreated 51 aa 51 aa 23 ab 520 ba 0.5 ab 10.2 ba 87 aba 107 aa 3.8 ba 0.2 bb Metam potassium 561 liters 75 aa 78 aa 10 cb 1,558 aa 0.1 bcb 20 aa 104 aa 74 bb 10.4 aa 0 bb Chloropicrin 263 kilograms 55 aa 54 aa 18 abb 430 ba 0.3 abb 8 ba 81 aba 54 bb 4.5 ba 0.1 bb Dimethyl disulfide 374 liters 61 aa 58 aa 4 db 15 da 0.1 bca 0.3 ca 36 ba 44 ba 9 aa 2.9 ab 1,3-dichloropropene 140 liters 65 aa 65 aa 10 cb 99 ca 0.2 bcb 1.5 bca 79 aba 53 bb 7.9 aa 0.5 bb 1,3-dichloropropene + chloropicrin 325 liters 124 aa 122 aa 14 bcb 57 ca 0.1 bca 0.5 ca 31 bb 65 ba 2.2 ba 1.1 ba Data are means of five replications on a split-plot design. Means within a column followed by a lowercase letter are not different based on Tukey s HSD test (P 0.05). Means within a row followed by an uppercase letter are not different according to Student s t-test (P 0.05). 1 Rate is broadcast equivalent. 2 Rf = reproductive factor (final J2 population density per 100 cm 3 of soil/ initial J2 population density per 100 cm 3 of soil). 100

101 Table 3-3. Residual effect of fumigant treatments and tomato cultivars in a double crop system where cucumber followed tomato. Rating 1 Marketable 2 Marketable weight 2 Galling index 3 (0-10) nu/ha kg/ha (0-100%) Treatments (rate/ha) 4 Nontreated 7.8 a 6,243 c (38) 1,328 c (40) 79 a Metam potassium 561 liters 8.2 a 10,118 a (0) 2,217 a (0) 39 c Chloropicrin 263 kilograms 7.7 a 6,243 c (38) 1,406 bc (37) 63 ab Dimethyl disulfide 374 liters 7.9 a 6,889 bc (32) 1,407 bc (36) 43 c 1,3-dichloropropene 140 liters 7.7 a 8,396 abc (17) 1,748 abc (21) 45 c 1,3-dichloropropene + chloropicrin 325 liters 8.2 a 8,611 ab (15) 2,012 ab (9) 32 c Tomato cultivars BHN a 7,987 a (0) 1,601 a (9) 71 a Crista 7.9 a 7,427 a (7) 1,767 a (0) 29 b Split-plot design with five replicates. Means within a column followed by a common letter are not different based on Tukey s HSD test (P 0.05). Parenthesis indicates percentage yield loss compared with the highest yield value. 1 Subjective plant vigor rating scale = 1 to 10, where 1 = small, chlorotic and non-vigorous plants 10 = large, dark green and vigorous plants. 2 Marketable yield = without malformation and 8 to 10 inches long. 3 Subjective root-knot nematode gall rating scale = 0 to 100, where 0 = no galls on the root system; 10 = 10% 100 = 100% of root system galled. 4 Rate is broadcast equivalent. 101

102 Table 3-4. Residual effect of treatment and tomato cultivars BHN 602 (Root-knot nematode susceptible) and Crista (Rootknot nematode resistant) on root-knot nematode population densities in soil and the reproductive factor in a double crop system where cucumber followed tomato. Tomato Cucumber Treatments (rate/ha) 1 Initial second-stage juvenile nu/100 cm 3 of soil Final second-stage juvenile nu/100 cm 3 of soil Rf 2 Final second-stage juvenile nu/100 cm 3 of soil Rf 2 Crista BHN 602 Crista BHN 602 Crista BHN 602 Crista BHN 602 Crista BHN 602 Nontreated 91 aa 92 aa 28 ab 407 aa 0.3 ab 4.4 aa 43 ab 123 aba 4.4 aa 0.3 cb Metam potassium 561 liters 101 aa 91 aa 24 aa 35 ba 0.2 aa 0.4 ba 42 ab 74 ba 0.4 cb 2.1 aa Chloropicrin 263 kilograms 97 aa 92 aa 25 ab 160 ba 0.3 ab 1.7 aba 19 ab 139 aa 1.7 ba 0.9 ba Dimethyl disulfide 374 liters 68 aa 68 aa 9 bb 39 ba 0.1 aa 0.6 ba 37 ab 71 ba 0.6 cb 1.8 aba 1,3-dichloropropene 140 liters 87 aa 83 aa 17 abb 36 ba 0.2 aa 0.4 ba 32 ab 95 aba 0.4 cb 2.6 aa 1,3-dichloropropene + chloropicrin 325 liters 57 aa 57 aa 17 abb 64bA 0.3 ab 1.1 aba 24 ab 72 ba 1.1 ba 1.1 ba Data are means of five replications on a split-plot design. Means within a column followed by a lowercase letter are not different based on Tukey s HSD test (P 0.05). Means within a row followed by an uppercase letter are not different according to Student s t-test (P 0.05). 1 Rate is broadcast equivalent. 2 Rf = reproductive factor (final J2 population density per 100 cm 3 of soil/ initial J2 population density per 100 cm 3 of soil. 102

103 CHAPTER 4 CONSTANT AND DIURNAL TEMPERATURE EFFECTS ON THE PENETRATION AND DEVELOPMENT OF ROOT-KNOT NEMATODES (M. arenaria, M. floridensis, and M. javanica) Introduction The efficacy of the root-knot resistant tomato varies with RKN species, tomato cultivars, and environmental conditions, and particularly soil temperature (Araujo et al., 1982a, 1982b; Devran et al., 2010; Verdejo-Lucas et al., 2013). Holtzman (1965) and Dropkin (1969) were the first to evaluate the effect of temperature in tomato containing the Mi gene. They demonstrated that resistance expression to RKN was lost at 28 C and above. With increasing temperature, there was less of a hypersensitive response to nematode infection. Since these initial reports others have investigated whether the basal temperature of 28 C would apply for different RKN species, populations, and tomato cultivars (Araujo et al., 1982a, b; Ammati et al., 1989; Abdul-Baki et al.,1996; Williamson, 1998; Devran et al., 2010; Verdejo-Lucas et al., 2013; and Carvalho et al., 2015). There has been considerable discrepancy in data published among these reports. There were reports of the complete loss of resistance at temperatures above 32 C (Dropkin, 1969; Williamson, 1998); whereas others showed that the Mi-gene was still effective in some cultivars at soil temperatures above 34 C (Abdul-Baki et al., 1996; Verdejo-Lucas et al., 2013). Abdul- Baki et al. (1996) mentioned that the resistant level of homozygous and heterozygous tomato genotypes was maintained fully at 31 C, partially maintained at 34 C, and lost at 37 C. They used an in vitro method and growth chamber studies whereby they were able to provide a growth pattern of isolated roots in a confined environment. Verdejo- Lucas et al. (2013) also conducted experiments under greenhouse conditions that 103

104 reached similar results. In soil temperatures fluctuating around 34 C resistance was still effective; however, it was not effective at a constant temperature of 34 C. During the growing season soil temperatures ranged from 9.7 to 34.1 C, and the number of days with soil temperatures above 28 C were 31 and the length of the heating period ranged from 0.5 to 6 hours per day. It appears that the effectiveness of the Mi-gene even at high soil temperatures was related with two main factors: soil temperatures at the beginning or at the end of growing season; and soil temperature fluctuations during the day (diurnal effect). Varying soil temperature and RKN inoculation periods showed that the Mi-gene resistance was lost after 4 days at temperatures above 33 C in 1-day-old seedlings, and subsequently placed at 27 C for 1 month (Dropkin, 1969). However, resistance was maintained when plants were held for 2 days at a temperature of 28 C before inoculation and subsequently held at 32 C after inoculation. It was concluded that the fate of J2 in tomato roots and effectiveness of the Mi-gene was determined in the first few hours after penetration (Dropkin et al., 1969). The Mi-gene effectiveness may recover from short periods of heat stress. Carvalho et al. (2015) showed that a single midday heat exposure to 35 C for 3 hours was sufficient to break Mi-gene resistance, thereby leading to a significant increase in gall formation; however, this increase in susceptibility did not persist and resistance was completely recovered after 6 days at 27 C. In addition, Zacheo et al. (1995) also reported that 6 days of heat at a constant temperature of 34 C resulted in seedlings becoming susceptible for 1 to 2 days, however when returned to a control temperature of 27 C they became resistant again. Interestingly, the return of resistance was 104

105 accompanied by an increase in peroxidase activity, which is associated with the hypersensitive response. This recovery of resistance could also explain why resistance remains effective when plants are under diurnal field temperatures. It is important to understand the length of time of heat exposure and temporal sequence of heat whereby the Mi-gene remains effective. It is also important to follow the different development stages of RKN in resistant and susceptible tomato cultivars, particularly, to determine the fate of J2 in a RKN resistant tomato cultivar after the resistance expression. The objectives were to: (1) evaluate different temperature regimes on the Migene response in tomato to infection by Meloidogyne arenaria, M. floridensis and M. javanica; and to (2) determine the fate of second-stage juveniles (J2) after the hypersensitive reaction occurs in a RKN resistant tomato root. To accomplish the objectives it was necessary to determine: i) the basal temperature for the breakdown of Mi-gene resistance; ii) the rate of development of Meloidogyne spp. in resistant and susceptible tomato cultivars held under constant temperatures and diurnal regimes; iii) the length of time to reach different developmental stages of growth at different temperatures; iv) the impact of multiple consecutive days of heat spikes in resistant tomato cultivars; and the susceptibility of infected plants to secondary nematode infection, and v) the reproductive ability of RKN species in resistant tomato cultivars. To accomplish the egression objective, it was necessary to quantify the number of J2 in soil and in root systems of resistant tomato cultivars and compare with the susceptible tomato cultivar. 105

106 Material and Methods Nematode Isolates and Inoculum Preparation Isolates of M. arenaria race 1, M. javanica race 2, and M. floridensis were maintained in a greenhouse at the Entomology and Nematology Department, University of Florida, Gainesville. Each species was derived from a single egg mass. Species and race identification was confirmed using perineal patterns, isozyme phenotypes, and differential host tests (Esbenshade and Triantaphyllou, 1990; Harris and Hopkinson, 1976; Hartman and Sasser, 1985). Eggs of the three nematodes species were extracted from galled tomato roots by immersing in 0.5% NaOCl solution (Bonetti and Ferraz, 1981). The eggs were caught on a sieve with 25-µm-pore openings and then placed on a modified aerated Baermann funnel (Rodriguez-Kabana and Pope, 1981) at approximately 27 C to hatch. Secondstage juveniles collected during the first 24 hours were discarded and the subsequent 48-hour-old J2 were used for the experiments. The concentration of J2 was estimated using a Peters counting slide (Peters, 1952). The volume of suspension was adjusted with tap water to obtain a concentration of 200 J2/ml. Each tomato seedling was inoculated with ca. 200 J2 by pipetting them into three 2.5-cm-deep-holes around the stem base. Plant Material Tomato seeds were germinated in a commercial growing mix (Fafard, BWI, Plymouth, FL) and maintained at 21 ± 8 ºC in a greenhouse. Two tomato cultivars were used: RKN resistant cv. Amelia and RKN susceptible cv. Agriset 344 (SeedWay, Hall, New York). 106

107 After the appearance of true leaves, around 3 to 4 weeks, the seedlings were transferred to small styrofoam cups (50 ml) containing builder s sand (97% sand and 3% clay). Before using the sand was placed in an autoclave at 250 ºC set at 124 KPa. Three holes were made in the bottom of each styrofoam cup with a dissecting needle to allow for drainage. The seedlings were held for 4 days before inoculation in environmental chambers (Percival Scientific, Inc. Perry, IA) at 25 ºC with 12 hours of light per day and a light intensity of 40 µmol/m -2 s -1. Heat Regime Experiments Four temperature regimes were evaluated: constant temperature of 28 and 32 ºC; diurnal temperatures of 26 ºC (12 h/night) and 32 ºC (12 h/day), and a constant temperature of 26 ºC for 3 days followed by diurnal temperatures of 26 ºC (12 h/night) and 32 ºC (12 h/day). Each nematode species was evaluated separately on different dates. After 72 hours exposure in the chamber, 40 tomato plants were removed from the sand and washed thoroughly with tap water to remove any J2 that had not penetrated (stopped penetration). The seedlings were then transplanted into sand media and placed again in the environmental chambers. Ten plants remained in the environmental chambers until females with egg masses appeared on the susceptible tomato cultivar (nonstopped penetration). Ten noninoculated plants remained in each chamber as a control. A total of 90 inoculated plants of each tomato cultivar were placed in each temperature regime. Seedlings were watered every other day with 10 ml of a fertilizer solution containing 0.21 g/liter of a soluble (N-P 2 O 5 -K 2 O) with micronutrients. 107

108 Data Collection Development of RKN species was determined by sequential sampling. For stopped penetration, three seedlings from each heat regime were harvested starting 5 days after inoculation (DAI) and then at 2 day intervals until the first appearance of females with egg masses. For nonstopped penetration, the seedlings were removed when egg laying females were observed. Noninoculated plants were harvested every 5 days. Roots of each plant were washed, cleared, and stained with acid fuchsin (Byrd et al., 1983). The roots were placed between two glass microscope slides (25 x 75 x 1 mm) and pressed. Developmental stages were observed with the aid of a stereo microscope (Discovery V12, Zeiss) at magnifications of x8 to x100 and identified according to the description of Triantaphyllou and Hirschmann (1960). When young females were observed, the plant root systems were first stained with 15 mg/liter Phloxine B solution to aid with the detection of egg masses (Dickson and Struble, 1965) before clearing and staining with acid fucshin. The number of J2, third-stage juveniles (J3), fourth-stage juveniles (J4), young females, and egg-laying females were recorded. For the nonstopped penetration seedlings the number of eggs per egg masses were quantified. At the first appearance of a hypersensitive reaction in the resistant tomato the number of J2 in the roots were counted. Any J2 that remained in the sand or had egressed from roots to the sand were recorded and counted by washing over a sieve with 25-µm-pore openings. 108

109 Statistical Analysis Statistical analysis was carried out with the non-parametric model procedure of SAS (SAS Institute, Cary, NC). Data on eggs per gram of root and reproductive factor were transformed by log 10 (x + 1) before analysis. Data from the tomato cultivars were compared and means separated based on Student s t-test. Results At the constant temperature of 28 C the Mi-1 gene remained functional for M. arenaria and M. javanica, but not for M. floridensis (Figures 4-1 to 4-9). For both M. arenaria (Figures 4-7 to 4-9) and M. javanica (Figures 4-4 to 4-6) in stopped and nonstopped penetration experiments only J2 were found at the end of the experiment in the resistant tomato cultivar. For all three species, the number of J2 recovered in the sand was greater in the resistant tomato cultivar treatment (Figures 4-2, 4-5, 4-8). Egg-laying females of M. floridensis were found in Amelia at day 25, whereas they were found in Agriset 344 at day 23 (Figure 4-1). In the nonstopped penetration experiment there were no differences among the different development stages except Amelia had lower values than Agriset 344 for J2 in soil and J3/J4 (Figures 4-3). At a constant temperature of 32 C egg-laying females developed for all three species (Figures 4-31 to 4-36). The species completed their life-cycle between 19 to 21 days (Figures 4-10 to 4-18, and 4-31 to 4-36). For M. arenaria and M. floridensis the life-cycle completion was delayed 4 days in Amelia when compared with Agriset 344 (Figures 4-10 and 4-16); whereas for M. javanica the life-cycle required the same time independently of the cultivar (Figures 4-13). Also, experiments with both stopped and nonstopped penetration of J2, males were found only in the resistant tomato cultivar infected with M. arenaria (Figures

110 and 4-18), and M. floridensis (Figures 4-11 and 4-12); whereas for M. javanica males were found only in the nonstopped penetration experiment and also in the resistant tomato cultivar (Figure 4-15). In the experiment with nonstopped penetration, M. arenaria had similar numbers of each development stage for both cultivars (Figure 4-15). With M. javanica only the number of J2 in soil was higher for Agriset compared with Amelia (Figure 4-12). With M. floridensis there were higher numbers of egg masses and galls per gram of roots in Agriset compared with Amelia (Figure 4-15). When evaluating the diurnal effect on the Mi-1 gene, there was no evidence of the loss of resistance for M. arenaria or M. javanica (Figures 4-19 to 4-29). For M. arenaria in the resistant tomato cultivar the latest developmental stage observed was one J3/J4 3-days following treatment at 26 C, then held at 26 C (12 hours) and 32 C (12 hours) for 25 days with stopped penetration (Figure 4-24). For M. javanica in the resistant tomato cultivar the latest developmental stage observed was one J3/J4 3-days following treatment at a constant temperature of 26, than held at 26 C (12 hours) and 32 C (12 hours) for 29 days with stopped penetration of J2 (Figure 4-20) and nonstopped penetration (Figure 4-21). Discussion Tomato with the Mi-1 gene continues to be the only available resistant gene for RKN in tomato (Mantelin et al., 2013). The use of this resistant gene under field conditions depends primarily in the ability to hold resistance against RKN at high soil temperatures and the appearance of RKN resistance breaking populations. Based on the field data reported in here (chapter two), the resistance to root-knot nematode was maintained even when temperatures reached above 32 and 34 C for 110

111 almost 24 hours, which is in agreement with previous studies (Dropkin, 1969; Verdejo- Lucas, 2013). Based on the soil temperature reported from field research (chapter two) this resistance loss would be expected in the fall season, where soil temperatures above 28 C at the beginning and middle of the growing season are common. In the spring growing season soil temperatures above 28 C occurred frequently during the middle and end of the growing season. Nevertheless, based on galling percentages obtained there was no evidence of this occurring. Improved understanding of how and when heat exposure disrupts Mi-1 resistance is essential to develop an efficient alternative to chemical management, especially with the suspension of methyl bromide. This research shows that heat units of 12 hours at 32 C or 28 C for 24 hours, were not enough to breakdown Mi-1 gene resistance but 32 C for 24 hours were. This suggest that maximum temperatures above 28 C were not as important as the duration of the heat units and this could explain the discrepancy among previous reports where there were different effects of temperature on the Mi-1 gene (Araujo et al., 1982a, b; Ammati et al., 1989; Abdul-Baki et al.,1996; Williamson, 1998; Devran et al., 2010; Verdejo-Lucas et al., 2013; and Carvalho et al., 2015). Our studies at a constant temperature of 32 C were in agreement with Zacheo et al., (1995) and Devran et al. (2010), where resistance to M. incognita broke down at temperatures of 32 C with heat treatments of at least 24 hours. This scenario of constant heat units above 28 C for 24 hours between 15 to 30 days, doesn t translate to the reality under field conditions. Based on temperature 111

112 graphics from chapter two there were daily temperature fluctuations. With high temperatures of at least 34 C for a period of 24 to 48 hours and lows of 19 C for a short period of time. Under these conditions there was maintenance of resistance, and these results are in agreement with previous studies (Verdejo-Lucas et al., 2013) were they found that Mi-1 resistance to M. arenaria and M. javanica remained intact in three of five cultivars exposed for 65 days to ambient soil temperatures as high as 34.1 C for more than 7.5 hours daily. The fact that exposed to diurnal temperatures were unable to cause loss of resistance might have been related with three factors: Mi-1 gene acclimated and recovered from exposure to heat units; time when heat units were applied; or the effect of low nocturnal temperatures. Carvalho et al. (2015) suggested that the Mi-1 gene can recover overtime from temperature peaks of 35 ºC for 3 hours during different periods of M. incognita infection. It was also mentioned that planting tomato during the hottest season should be avoided, but occasional short-term high temperatures in the field will likely not greatly increase galling by M. incognita. Based on our study it was also possible for RKN resistant tomato cultivar recovered from heat peaks of 32 ºC for 12 hours since there was no formation of galls or any developmental stage in the root system beside J2. This ability to recover might also be related with low temperatures. Araujo et al (1982a, b) found that plants exposed to low temperatures of 19 to 25 ºC and then high diurnal temperature of 31 ºC were better able to maintain resistance than plants exposed to temperatures lower or higher than 28 ºC. However, this was not seen in this study at constant temperature of 28 ºC where the resistant was still functional. 112

113 Nevertheless, more research should be done to understand really what is the basal temperature for a significant nonfunctioning of the Mi-1 gene. The time at which heat units were applied also might have influence, therefore in this study the diurnal effects tested in two different regimes showed that the J2 were allowed to penetrate and try to establish a feeding site at the constant temperature of 26 ºC for 3 days followed by temperature fluctuation regime; and a second experiment where after inoculation of J2 until the end of life-cycle temperature fluctuations were applied. In both experiences, there was no evidence that temperature had an effect on the RKN life-cycle and resistance. Although, the recovery of Mi-1 gene after short-time heat spikes might explain the reason why diurnal effect was unable to result in the loss of resistance; we should keep in consideration the duration of the heat unit more than the high temperature itself. More work should be done to evaluate the time needed to break resistance. The RKN species applied has influence on the Mi-gene performance. Some virulent species, namely M. enterolobii and M. floridensis are reported to break resistant genes in several species including tomato (Brito et al., 2007a, 2007b). Both species are reported in Florida and are a potential problem for crop production. As shown in this study, independently of temperature, M. floridensis was able to overcome Mi-1 resistance; although there was a delay in the life-cycle of 4 days between RKN resistance and susceptible tomato cultivar held at a constant temperature of 32 ºC. In terms of possible egression from tomato roots it was clear that higher numbers of J2 were collected from the three RKN species in soil and roots in RKN resistant tomato cultivars than in a susceptible cultivar. Finding J2 in the soil in experiments with 113

114 stopped penetration indicated that some J2 egressed from the roots, whereas others were not able to do so, probably due to the lack of stored energy. The ability to egress from roots might not only be related with the lack of energy but also lack of cues from other root systems. In the experiment of nonstopped penetration at 26 C (12 hours) and 32 C (12 hours) for 25 days for M. javanica, J2 were found in the soil and root of the RKN susceptible tomato cultivar. This is related with the fact that we had the beginning of a second life-cycle. In general, cultivars with the Mi-1 gene were not immune to RKN infection. The fact that in some RKN resistant cultivars we were able to find late stage J2 and a few J3/J4 indicates that some individuals are able to suppress plant resistance defenses and continue their life-cycle even if it means a delay of several days when compared with RKN susceptible tomato cultivars. This research was unique, not because of the studies of diurnal effect but of the ability to show different developmental stages of RKN in the roots. Previous studies based their results in values of galling percentages that are important; however they can be highly subjective since the presence of galls don t really represent the reproduction of RKN species in cultivars with Mi-1 gene. We were able to show the effect of Mi-1 gene in the perspective of RKN development. More work needs to be done on the effects of diurnal temperatures, this is an important factor understanding why the Mi-1 gene works under field conditions with high soil temperatures. Again, more than the maximum temperatures applied it would be important to understand the time required for heat to influence the functioning of Mi-1 gene. Zhu et al., (2010), suggested that heat-associated breakdowns in Mi-gene 114

115 resistance was affected by changes downstream of gene expression, such as protein conformational changes. It would be interesting to understand the duration of heat units able to create dysfunctional proteins. 115

116 Number of each root-knot nematode developmental stage 60 A J2 in soil J2 J3/J4 B Female without eggs Female with eggs Days after inoculation Figure 4-1. Mean number of Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days at a constant temperature of 28 C. Penetration of second-stage juveniles was stopped 3 days after inoculation. Mean of three replicates. 116

117 Number of each root-knot nematode developmental stage * * RKN resistant tomato cultivar RKN susceptible tomato cultivar * J2 in soil J2 Late J2 J3/J4 Female Female with without eggs eggs Males Figure 4-2. Total number of each Meloidogyne floridensis developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato held 25 days at constant temperature of 28 C. Penetration of second-stage juveniles was stopped after 72 hours. * Indicates difference based on Student s t-test (P 0.05). 117

118 Number of each root-knot nematode developmental stage * RKN susceptible tomato cultivar RKN resistant tomato cultivar * * * J2 in soil J2 in roots J3/J4 Females Females with egg masses Eggs/eggmass Males Number egg Number galls/ g masses/ g fresh fresh root root Figure 4-3. Mean number of Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held 25 days at constant temperature of 28 C. Penetration of second-stage juveniles was not stopped 3 days after inoculation. Mean of 10 replicates.* Indicates difference based on Student s t-test (P 0.05).. 118

119 Number of each root-knot nematode developmental stage 60 A J2 in soil J2 J3/J4 Female without eggs Female with eggs B Days after inoculation Figure 4-4. Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days at a constant temperature of 28 C. Penetration of second-stage juveniles was stopped 3 days after inoculation. Mean of three replicates. 119

120 Number of each root-knot nematode developmental stage * RKN resistant tomato cultivar RKN susceptible tomato cultivar 150 * 100 * 50 0 J2 in soil J2 Late J2 J3/J4 Female Female with without eggs eggs * * Males Figure 4-5. Total number of each Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at constant temperature of 28 C. Penetration of second-stage juveniles was stopped after 72 hours. * Indicates difference based on Student s t-test (P 0.05). 120

121 300 RKN susceptible tomato cultivar 250 * RKN resistant tomato cultivar * 100 * 50 0 * * J2 in soil J2 in roots J3/J4 Females Females with egg masses * Eggs/eggmasses Males Number of egg masses/ g fresh root * Number of galls/ g fresh root Figure 4-6. Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at constant temperature of 28 C. Penetration of second-stage juveniles was not stopped 3 days after inoculation. Mean of 10 replicates.* Indicates difference based on Student s t-test (P 0.05). 121

122 Number of each root-knot nematode developmental stage A J2 in soil J2 J3/J4 B Female without eggs Female with eggs Days after inoculation Figure 4-7. Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days at a constant temperature of 28 C. Penetration of second-stage juveniles was stopped 3 days after inoculation. Mean of three replicates. 122

123 Number of each root-knot nematode developmental stage * RKN resistant tomato cultivar RKN susceptible tomato cultivar * * * J2 in soil J2 Late J2 J3/J4 Female Female with without eggs eggs * * Males Figure 4-8. Total number of each Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at constant temperature of 28 C. Penetration of second-stage juveniles was stopped after 72 hours. * Indicates difference based on Student s t-test (P 0.05). 123

124 Number of each root-knot nematode developmental stage RKN susceptible tomato cultivar 250 * RKN resistant tomato cultivar * 50 0 * * J2 in the soil J2 in the roots J3/J4 Females Females with egg masses Eggs/eggmass Males Number of egg masses/ g fresh root * Number of galls/ g fresh root Figure 4-9. Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at constant temperature of 28 C. Penetration of second-stage juveniles was not stopped 3 days after inoculation. Mean of 10 replicates.* Indicates difference based on Student s t-test (P 0.05). 124

125 Number of each root-knot nematode developmental stage 25 A B J2 in soil J2 J3/J4 Female without eggs Days after inoculation Figure Mean number of Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 15 and19 days at a constant temperature of 32 C. Penetration of second-stage juveniles was stopped 3 days after inoculation. Mean of three replicates.. 125

126 Number of each root-knot nematode developmental stage * * * * RKN resistant tomato cultivar RKN susceptible tomato cultivar J2 in soil J2 Late J2 J3/J4 Female Female with without eggs eggs Males Figure Total number of each Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 15 and 19 days at constant temperature of 32 C. Penetration of second-stage juveniles was stopped after 72 hours. * indicates difference based on Student s t-test (P 0.05). 126

127 Number of each root-knot nematode developmental stage RKN susceptible tomato cultivar 180 RKN resistant tomato cultivar * J2 in the soil J2 in the roots J3/J4 females Females with egg masses Eggs/eggmass Males Number egg masses/ g fresh root Number of galls/ g fresh root Figure Mean number of Meloidogyne floridensis developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 19 days at constant temperature of 32 C. Penetration of second-stage juveniles was not stopped 3 days after inoculation. Mean of 10 replicates.* Indicates difference based on Student s t-test (P 0.05). 127

128 Number of each root-knot nematode developmental stage 30 A J2 in soil J2 B J3/J Female without eggs Female with eggs * Days after inoculation Figure Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 19 days at a constant temperature of 32 C. Penetration of second-stage juveniles was stopped 3 days after inoculation. Mean of three replicates. 128

129 Number of each root-knot nematode developmental stage RKN resistant tomato cultivar 120 RKN susceptible tomato cultivar 100 * * J2 in soil J2 Late J2 J3/J4 Female Female with without eggs eggs Males Figure Total number of each Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held 19 days at constant temperature of 32 C. Penetration of second-stage juveniles was stopped after 72 hours. * indicates difference based on Student s t-test (P 0.05). 129

130 Number of each root-knot nematode developmental stage RKN susceptible tomato cultivar RKN resistant tomato cultivar * * * 20 0 J2 in the soil J2 in the roots J3/J4 Female Female with egg masses Eggs/eggmass Males Number of galls Number of egg Number of egg Number of masses masses per grams of fresh root galls per gram of fresh root Figure Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 19 days at constant temperature of 32 C. Penetration of second-stage juveniles was not stopped 3 days after inoculation. Mean of 10 replicates.* Indicates difference based on Student s t-test (P 0.05) 130

131 Number of each root-knot nematode developmental stage A J2 in soil J2 B J3/J4 Female without eggs Female with eggs * Days after inoculation Figure Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 19 and 21 days at a constant temperature of 32 C. Penetration of second-stage juveniles was stopped 3 days after inoculation. Mean of three replicates. 131

132 Number of each root-knot nematode developmental stage * RKN resistant tomato cultivar RKN susceptible tomato cultivar * * J2 in soil J2 Late J2 J3/J4 Female Female with without eggs eggs Males Figure Total number of each Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivar held for 21 days at constant temperature of 32 C. Penetration of second-stage juveniles was stopped after 72 hours. * Indicates difference based on Student s t-test (P 0.05). 132

133 Number of each root-knot nematode developmental stage RKN susceptible tomato cultivar RKN resistant tomato cultivar J2 in soil J2 in roots J3/J4 Females Female with egg masse Eggs/eggmass Number of galls Number of eggnumber of egg masses masses/ g fresh root Number of galls/ g fresh root Figure Mean number of Meloidogyne arenaria developmental stages observed in roots of for root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 21 days at constant temperature of 32 C. Penetration of second-stage juveniles was not stopped 3 days after inoculation. Mean of 10 replicates.* Indicates difference based on Student s t-test (P 0.05). 133

134 Number of each root-knot nematode developmental stage A B J2 J3/J4 Female without eggs Female with eggs Days after inoculation Figure Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 29 days at 3 days at 26 C followed by 26 C (12 hours) and 32 C (12 hours). Penetration of second-stage juvenile was stopped 3 days after inoculation. Mean of three replicates. 134

135 Number of each root-knot nematode developmental stage RKN resistant tomato cultivar RKN susceptible tomato cultivar * * * * 50 * 0 J2 in soil J2 Late J2 J3/J4 Female Female with without eggs eggs Males Figure Total number of each Meloidogyne javanica developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 29 days at 3 days at 26 C followed by 26 C (12 hours) and 32 C (12 hours). * Indicates difference based on Student s t- test (P 0.05). 135

136 Number of each root-knot nematode developmental stage RKN susceptible tomato cultivar * RKN resistant tomato cultivar * * * * 0 J2 in soil J2 in roots J3/J4 Females Female with egg masses Eggs/eggmass Males Number of egg masses/ g fresh root Number of galls/ g fresh root Figure Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 29 days at 3 days at 26 C followed by 26 C (12 hours) and 32 C (12 hours). Mean of 10 replicates. * Indicated difference based on Student s t-test (P 0.05). 136

137 Number of each root-knot nematode developmental stage A J2 J3/J4 Female without eggs Female with eggs B Days after inoculation Figure Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 29 days at 3 days at 26 C followed by 26 C (12 hours) and 32 C (12 hours). Penetration of second-stage juvenile was stopped 3 days after inoculation. Mean of three replicates. 137

138 Number of each root-knot nematode developmental stage * RKN resistant tomato cultivar RKN susceptible tomato cultivar * * * * 50 * 0 J2 in soil J2 Late J2 J3/J4 Female Female with without eggs eggs Males Figure Total number of each Meloidogyne arenaria developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 29 days at 3 days at 26 C followed by 26 C (12 hours) and 32 C (12 hours). * indicates difference based on Student s t- test (P 0.05). 138

139 Number of each root-knot nematode developmental stage * RKN susceptible tomato cultivar RKN resistant tomato cultivar * * * * * 0 J2 in soil J2 in roots J3/J4 Females Female with egg mass Eggs/eggmass Males Number of egg Number of galls masses/ g fresh / g fresh root root Figure Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 29 days at 3 days at 26 C followed by 26 C (12 hours) and 32 C (12 hours). Mean of 10 replicates. * Indicated difference based on Student s t-test (P 0.05). 139

140 Number of each root-knot nematode developmental stage 45 A J2 in soil B J2 J3/J4 Female without eggs Female with eggs * Days after inoculation Figure Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days at 26 C (12 hours) and 32 C (12 hours). Penetration of second-stage juvenile was stopped 3 days after inoculation. Mean of three replicates. 140

141 Number of each root-knot nematode developmental stage RKN resistant tomato cultivar * RKN susceptible tomato cultivar * 150 * * J2 in soil J2 Late J2 J3/J4 Female Female with without eggs eggs * * Males Figure Total number of each Meloidogyne javanica developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at 26 C (12 hours) and 32 C (12 hours). * indicates difference based on Student s t-test (P 0.05). 141

142 Number of each root-knot nematode developmental stage * RKN susceptible tomato cultivar RKN resistant tomato cultivar * * * * 0 J2 in soil J2 in roots J3/J4 Females Female with egg mass Eggs/eggmass Males Number of egg masses/ g fresh root Number of galls/g fresh root Figure Mean number of Meloidogyne javanica developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at 26 C (12 hours) and 32 C (12 hours). Mean of 10 replicates. * Indicated difference based on Student s t-test (P 0.05). 142

143 Number of each root-knot nematode developmental stage 70 A J2 J3/J4 Female without eggs Female with eggs B Days after inoculation Figure Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant A) Amelia, and susceptible B) Agriset 344, tomato cultivars held for 25 days at 26 C (12 hours) and 32 C (12 hours). Penetration of second-stage juvenile was stopped 3 days after inoculation. Mean of three replicates. 143

144 Number of each root-knot nematode developmental stage 350 RKN resistant tomato cultivar 300 RKN susceptible tomato cultivar * * J2 in soil J2 Late J2 J3/J4 Female without eggs * * * Female with eggs Males Figure Total number of each Meloidogyne arenaria developmental stage observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at 26 C (12 hours) and 32 C (12 hours) for 25 days. * indicates difference based on Student s t-test (P 0.05). 144

145 Number of each root-knot nematode developmental stage 250 * RKN susceptible tomato cultivar RKN resistant tomato cultivar * * J2 in soil J2 in roots J3/J4 Females Female with egg mass * * * Eggs/eggmass Males Number of egg/ g fresh root Number of galls/ g fresh root Figure Mean number of Meloidogyne arenaria developmental stages observed in roots of root-knot nematode resistant (Amelia) and susceptible (Agriset 344) tomato cultivars held for 25 days at 26 C (12 hours) and 32 C (12 hours). Mean of 10 replicates. * Indicated difference based on Student s t-test (P 0.05). 145

146 A B C D Figure Developmental stages of Meloidogyne floridensis in Agriset 344 roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=late J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4); D=laying egg female). Courtesy of author. 146

147 A B C D Figure Developmental stages of Meloidogyne floridensis in Amelia roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=late J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4); D=laying egg female). Courtesy of author. 147

148 A B C D Figure Developmental stages of Meloidogyne arenaria in Agriset 344 roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=late J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4); D=laying egg female). Courtesy of author. 148

149 A B C D Figure Developmental stages of Meloidogyne arenaria in Amelia roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=J2 egressing from root; C=third-stage juvenile (J3)/fourth-stage juvenile (J4); D=laying egg female). Courtesy of author. 149

150 A B C D Figure Developmental stages of Meloidogyne javanica in Agriset 344 roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4); D=laying egg female). Courtesy of author. 150

151 A B C D Figure Developmental stages of Meloidogyne javanica in Amelia roots held at a constant temperature of 32 C. (A= second-stage (J2) infective juvenile; B=late J2; C=third-stage juvenile (J3)/fourth-stage juvenile (J4); D=laying egg female). Courtesy of author. 151